Cd40 targeted peptides and uses thereof

ABSTRACT

The present disclosure is related to compositions comprising peptides that bind CD40 and methods of use in inhibiting interaction of CD40 and CD154 and inducing immunosuppression. Provided herein are methods of transplantation and methods of inhibiting donor specific immune response. Also provided herein are methods of treatment for autoimmune diseases, inflammatory diseases and cancer.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.17/089,463, filed Nov. 4, 2020, which is a continuation of InternationalPatent Application PCT/US2019/061325, filed Nov. 13, 2019, which claimsbenefit to U.S. Provisional Patent Application No. 62/760,796, filedNov. 13, 2018, each of which is incorporated herein by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 13, 2021, isnamed 199830-718302-Squence_Listing.txt and is 8,192 bytes in size.

BACKGROUND OF THE DISCLOSURE

CD40 ligand (CD40L), or CD154, binds to i) its classical receptor CD40on B cells and other antigen presenting cells, ii) αMβ2, also known asMac-1, on neutrophils, monocytes, and macrophages, iii) α5β1 onmonocytes, and αIIbβ3 on platelets. The interaction of CD154 with CD40is implicated in the activation of CD40 expressing immune cells such asB-cell, T-cell, neutrophil, monocyte, macrophage, dendritic cell, andplatelet.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.Absent any indication otherwise, publications, patents, and patentapplications mentioned in this specification are incorporated herein byreference in their entireties.

SUMMARY OF THE DISCLOSURE

In one aspect provided herein is a pharmaceutical composition comprisinga peptide or a portion thereof, wherein the peptide comprises an aminoacid sequence with at least 85%, or at least 90% sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NOs:1-6 and 8-17, and a pharmaceutically acceptable carrier, wherein saidpeptide binds a CD40 protein or portion thereof. In one aspect, providedherein is a pharmaceutical composition comprising a peptide, wherein thepeptide comprises an amino acid sequence with at least 90% sequenceidentity to the sequences set forth in any one of SEQ ID NOs: 1-6 and8-17, and a pharmaceutically acceptable carrier, wherein said peptidebinds a CD40 protein or a portion thereof.

In some embodiments, the peptide comprises the amino acid sequence ofSEQ ID NO: 1. In some embodiments, the peptide comprises the amino acidsequence of SEQ ID NO: 2. In some embodiments, the peptide comprises theamino acid sequence of SEQ ID NO: 3. In some embodiments, the peptidecomprises the amino acid sequence of SEQ ID NO: 4. In some embodiments,the peptide comprises the amino acid sequence of SEQ ID NO: 5. In someembodiments, the peptide comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the peptide comprises the amino acid sequence ofSEQ ID NO: 8. In some embodiments, the peptide comprises the amino acidsequence of SEQ ID NO: 9. In some embodiments, the peptide comprises theamino acid sequence of SEQ ID NO: 10. In some embodiments, the peptidecomprises the amino acid sequence of SEQ ID NO: 11. In some embodiments,the peptide comprises the amino acid sequence of SEQ ID NO: 12. In someembodiments, the peptide comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, the peptide comprises the amino acid sequenceof SEQ ID NO: 14. In some embodiments, the peptide comprises the aminoacid sequence of SEQ ID NO: 15. In some embodiments, the peptidecomprises the amino acid sequence of SEQ ID NO: 16. In some embodiments,the peptide comprises the amino acid sequence of SEQ ID NO: 17.

In some embodiments, the peptide is from 4 to 20 amino acids in length.In some embodiments, the peptide comprises at least one non-naturallyoccurring amino acid. In some embodiments, the peptide comprises acarboxy terminus that is amidated. In some embodiments, the peptide isin a cyclic form. In some embodiments, the peptide further comprisesPEG. In some embodiments, the peptide is comprises a lipophilicmolecular group. In some embodiments, the peptide is recombinant orsynthesized. In some embodiments, the peptide is fused to a carrierpolypeptide. In some embodiments, the peptide is further conjugated to adetectable agent, a peptide tag, a magnetic particle, a diagnosticagent, a therapeutic agent, or a combination thereof. In someembodiments, the pharmaceutical composition disclosed herein, comprisesa detectable agent that is an enzyme—substrate agent, a fluorescentagent, a chemiluminescent agent, or a radioisotope. In some embodiments,the pharmaceutical composition disclosed herein, comprises a therapeuticagent that is an anti-inflammatory, immunosuppressive, immunomodulatory,or an anti-tumor agent. In some embodiments, the peptide is in a solubleform.

In some embodiments, the peptide is formulated in a liposome or ananoparticle delivery system. In some embodiments, the peptide isformulated with a biocompatible polymer. In some embodiments, thepharmaceutical composition of the aspects above further comprises apharmaceutically acceptable diluent, additive or excipient. In someembodiments, the pharmaceutical composition of the aspects disclosedabove is formulated for administration via a subcutaneous, intravenous,intradermal, intraperitoneal, oral, intramuscular,intracerebroventricular, intranasal, intracranial, intracelial,intracerebellar, intrathecal, transdermal, pulmonary, or topicaladministration route. In some embodiments, the pharmaceuticalcomposition of the aspects disclosed above is formulated foradministration via intravenous administration route. In someembodiments, the pharmaceutical composition further comprises aganglioside and/or a phosphotidylserine.

In some embodiments, the pharmaceutical composition further comprisessaccharides selected from the group consisting of cyclodextrins,disaccharides, polysaccharides, and a combination thereof. In someembodiments, the pharmaceutical composition of the aspects disclosedabove is in a solution form or in a lyophilized form. In someembodiments, the pharmaceutical composition is contained in a deliverydevice selected from the group consisting of a syringe, a blunt tipsyringe, a catheter, an inhaler, a nebulizer, a nasal spray pump, anasal irrigation pump or nasal lavage pump, and an implantable pump. Insome embodiments, the pharmaceutical composition has a shelf life of atleast 2 days, 2 weeks, 1 month to 2 years at room temperature. In someembodiments, the pharmaceutical composition has a shelf life of at least2 days, 2 weeks, 1 month to 2 years at 4° C. In some embodiments, thepeptide or portion thereof binds a CD40 protein or a portion of the CD40protein. In some embodiments, the peptide or portion thereof, whereinthe peptide or the functional thereof is in an amount sufficient toinhibit binding of the CD40 protein to a CD154.

In one aspect, the technology herein relates to a complex comprising apeptide or a portion thereof non-covalently bound to a CD40 protein or aportion of the CD40 protein, wherein the peptide comprises an amino acidsequence with at least 85% or at least 90% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-6 and8-17. In some embodiments, the CD40 protein is mammalian. In someembodiments, the CD40 protein is human. In some embodiments, the CD40protein is recombinant. In one aspect provided herein is an isolatednucleic acid fragment encoding a peptide or a portion thereof, whereinthe peptide comprises an amino acid sequence with at least 85% or atleast 90% sequence identity to an amino acid sequence set forth in anyone of SEQ ID NOs: 1-6 and 8-17. In some embodiments, the isolatednucleic acid fragment of the aspects disclosed above further comprisinga nucleotide sequence encoding an agent, wherein the agent is atargeting agent, a detectable agent, a diagnostic agent, or atherapeutic agent. In one aspect provided herein is a vector comprisingthe isolated nucleic acid fragment of any one of aspects disclosedabove. In some embodiments, the isolated nucleic acid fragment isoperably linked to a regulatory control sequence. In one aspect providedherein is a host cell comprising the isolated nucleic acid fragment ofaspects above or the vector of aspects above.

In one aspect, provided herein is a preparatory regimen fortransplantation of a cell, tissue or organ from a recipient to a donor,comprising an anti-CD40 agent; wherein the anti-CD40 agent comprises apeptide containing an amino acid sequence with at least 85% or at least90% sequence identity to the sequences set forth in any one of SEQ IDNOs. 1-6 and 8-17. In another aspect, provided herein is a preparatoryregimen for transplantation of a cell, tissue or organ from a recipientto a donor, comprising an anti-CD40 agent; wherein the anti-CD40 agentcomprises a peptide containing an amino acid sequence with at least 90%sequence identity to the sequences set forth in any one of SEQ ID NOs.8-17. In one embodiment, the preparatory regimen can further compriseinstructions for administration of said anti-CD40 agent to saidrecipient. In one embodiment, said anti-CD40 agent is administered tosaid recipient between about 10 days before and about 30 days after saidtransplantation. In another embodiment, said anti-CD40 agent isadministered to said recipient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10days before said transplantation. In another embodiment, said anti-CD40agent is administered to said recipient at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25days before said transplantation. In another embodiment, said anti-CD40agent is administered to said recipient subcutaneously, intravenously,intradermally, intraperitoneally, orally, intramuscularly,intracerebroventricularly, intranasally, intracranially, intracelially,intracerebellarly, intrathecally, or transdermally, or topically. Inanother embodiment, said anti-CD40 agent is administered to saidrecipient at a dose of between about 1 mg/kg and 100 mg/kg. In oneembodiment, the anti-CD40 agent is administered to said recipient at adose of 50 mg/kg.

In one embodiment, the preparatory regimen provides long term toleranceto said transplant cell, tissue, or organ. In one embodiment, thepreparatory regimen further comprises administering an effective amountof apoptotic donor leukocytes. In one embodiment, the donor leukocytesare fixed in a cross-linking agent. In one embodiment, the apoptoticleukocytes are mammalian leukocytes. In one embodiment, the apoptoticleukocytes are porcine leukocytes. In one embodiment, the apoptoticleukocytes are human leukocytes. In one embodiment, the apoptoticleukocytes are from a cadaveric donor, a brain dead donor, a non-heartbeating donor, or a living donor. In one embodiment, the apoptoticleukocytes are ex vivo expanded leukocytes. In one embodiment, theapoptotic leukocytes are isolated from a spleen, or peripheral blood. Inone embodiment, the apoptotic leukocytes comprise B-lymphocytes. In oneembodiment, the apoptotic leukocytes comprise cells that have beendifferentiated from stem cells or induced pluripotent stem cells exvivo. In some embodiments, the stem cells are derived from the donor ofsaid transplant cell, tissue, or organ. In one embodiment, the apoptoticleukocytes and the recipient are matched for at least one of MHC class IA allele, MHC class I B allele, MHC class II DR allele, MHC class II DQallele, or MHC class II DP allele. In one embodiment, the apoptoticleukocytes and the transplant are matched for at least one of MHC classI A allele, MHC class I B allele, MHC class II DR allele, MHC class IIDQ allele, or MHC class II DP allele. In one embodiment, the apoptoticleucocytes and the transplant are haploidentical. the apoptoticleucocytes are from the donor of the transplant.

In one embodiment, transplant cell, tissue, or organ is a kidney, liver,heart, lung, pancreas, islet cell, small bowel, bone marrow,hematopoietic stem cell, embryonic stem cell-derived islet beta cell,induced pluripotent stem cell-derived islet beta cell, embryonic stemcell-derived islet, induced pluripotent stem cell-derived islet, a stemcell derived cell, tissue or organ, or a combination thereof. In oneembodiment, recipient and the donor of said transplant cell, tissue, ororgan are matched for at least one MEW class I A allele, MEW class I Ballele, MHC class II DR allele, MEW class II DQ allele, or MHC class IIDP allele.

In one aspect, provided herein is a preparatory regimen fortransplanting a cell, tissue or organ transplant to a recipient,comprising: one or more anti-CD40 peptides conjugated to the surface ofan apoptotic leukocyte, wherein the one or more anti-CD40 peptides,comprise an amino acid sequence with at least 85% or at least 90%sequence identity to a sequence selected from SEQ ID NOs: 1-6 and 8-17.In another aspect, provided herein is a preparatory regimen fortransplanting a cell, tissue or organ transplant to a recipient,comprising: one or more anti-CD40 peptides conjugated to the surface ofan apoptotic leukocyte, wherein the one or more anti-CD40 peptides,comprises an amino acid sequence with at least 90% sequence identity toa sequence selected from SEQ ID NOs: 8-17. the apoptotic leukocyte isfurther conjugated to one or more peptides derived from a MEW class IImolecule of the recipient. In one embodiment, the apoptotic leukocyte isfurther conjugated to one or more peptides derived from a MEW class IImolecule of the recipient. In one embodiment, the one or more peptidesderived from the MHC class II molecule comprises peptides derived from aDR β-chain, a DQ β-chain, or a DP β-chain, or a combination thereof. Inone embodiment, the one or more peptides derived from the MHC class IImolecule comprise a sequence from a hypervariable region. In oneembodiment, the one or more peptides conjugated to the surface of theleukocyte are synthesized in-vitro or recombinantly produced. In oneembodiment, the apoptotic leukocyte is MHC class II matched to the donorand MEW class II mismatched to the recipient. In one embodiment, theapoptotic leukocyte is conjugated to one or more peptides derived from aMEW class I molecule of the donor of the cell, tissue or organtransplant. In one embodiment, the MEW class I molecule is expressed inthe donor of the transplant. In one embodiment, the MHC class I moleculeis HLA-A1, HLA-A3, HLA-B7, or HLAB8. In one embodiment, the MEW class Imolecule is encoded by HLA-A*02, 24, 01 or HLA-B*35, 44, 51. In oneembodiment, the apoptotic leukocyte is MEW class I matched to therecipient, or MHC class II matched to the recipient, or both.

In one embodiment, the preparatory regimen further comprisesadministering one or more agents for short-term immunosuppression ofsaid recipient. In one embodiment, the one or more agents for short termimmunosuppression is an anti-CD40 agent, wherein the anti-CD40 agentcomprises a peptide containing an amino acid sequence with at least 85%or at least 90% sequence identity to an amino acid sequence set forth inany one of SEQ ID NOs: 1-6 and 8-17. In one embodiment, the one or moreagents for short term immunosuppression is an anti-CD40 agent, whereinthe anti-CD40 agent comprises a peptide containing an amino acidsequence with at least 90% sequence identity to an amino acid sequenceset forth in any one of SEQ ID NOs: 8-17. In one embodiment, the one ormore agents for short term immunosuppression comprise an mTOR inhibitor,an anti-tumor necrosis factor agent or an anti-tumor necrosis factorreceptor agent, an anti-interleukin 6 agent or an anti-interleukin 6receptor agent, an anti-CD40 agent, or a combination thereof. In oneembodiment, the preparatory regimen further comprises instructions foradministration of said one or more agents for short termimmunosuppression to said transplant recipient. In one embodiment, theapoptotic leukocytes have further been contacted with an amount of oneor more immunomodulatory molecules. In one embodiment, said amount ofone or more immunomodulatory molecules is sufficient to modify afunction of antigen-presenting cells in said recipient. In oneembodiment, said one or more immunomodulatory molecules are selectedfrom IFN-γ, an NF-kB inhibitor, vitamin D3, siCD40, cobaltprotoporphyrin, insulin B9-23, all or a portion of a cluster ofdifferentiation protein, or a combination thereof. In one embodiment,said NF-kB inhibitor is curcumin, triptolide, Bay-117085, or acombination thereof.

In one embodiment, the one or more peptides are conjugated to thesurface of the apoptotic leukocytes by treatment with the crosslinkingagent. In one embodiment, said crosslinking agent comprises acarbodiimide. In one embodiment, said carbodiimide comprises1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI);N,N′-diisopropylcarbodiimide (DIC); N,N′-dicyclohexylcarbodiimide (DCC);or a combination thereof. In one embodiment, said crosslinking agentdoes not comprise a carbodiimide. In one embodiment, said crosslinkingagent comprises genipin, acrylic aldehyde, diformyl, osmium tetroxide, adiimidoester, mercuric chloride, zinc sulphate, zinc chloride,trinitrophenol (picric acid), potassium dichromate, ethanol, methanol,acetone, acetic acid, or a combination thereof. IN one embodiment, saiddiimidoester is selected from cyanuric chloride, diisocyanate,diethylpyrocarbonate (DEPC), a maleimide, benzoquinone, or a combinationthereof. In one embodiment, said apoptotic leukocytes are fixed for apredetermined amount of time. In one embodiment, said predetermined timeis at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50minutes, 60 minutes, 75, minutes, 90 minutes, 120 minutes, 150 minutes,180 minutes, 210 minutes, or 240 minutes. In one embodiment, saidapoptotic leukocytes are contacted with an amount of one or moreimmunomodulatory molecules, before fixation.

In one aspect, provided herein is a tolerizing regimen forpost-transplant stabilization of a cell, tissue or organ transplant in arecipient, comprising administering to the recipient an effective amountof an anti-CD40 peptide, wherein the anti-CD40 peptide comprises anamino acid sequence with at least 85%. or at least 90% sequence identityto a sequence selected from SEQ ID NOs. 1-6 and 8-17. In another aspect,provided herein is a tolerizing regimen for post-transplantstabilization of cell, tissue or organ transplant in a recipient,comprising administering to the recipient an effective amount of ananti-CD40 peptide, wherein the anti-CD40 peptide comprises an amino acidsequence with at least 90% sequence identity to a sequence selected fromSEQ ID NOs. 8-17. In one embodiment, the tolerizing regimen furthercomprises apoptotic leukocytes. In one embodiment, the apoptoticleukocytes are expanded at least about 3 fold, 5 fold, 10 fold, 50 fold,100 fold, 150 fold, 200 fold or 250 fold relative to the startingpopulation prior to contacting with said crosslinking agent. In oneembodiment, the apoptotic leukocytes and the anti-CD40 peptide areadministered separately. In one embodiment, the apoptotic leukocytes andthe anti-CD40 peptide are administered simultaneously.

In one aspect, provided herein is a method of inhibiting an interactionbetween a CD40 protein and a CD154 protein, the method comprisingcontacting the CD40 protein with an anti-CD40 agent, wherein theanti-CD40 agent comprises a peptide containing an amino acid sequencewith at least 85% or at least 90% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-6 and 8-17,thereby inhibiting the interaction between the CD40 protein and theCD154 protein. In another aspect, provided herein is a method ofinhibiting an interaction between a CD40 protein and a CD154 protein,the method comprising contacting the CD40 protein with an anti-CD40agent, wherein the anti-CD40 agent comprises a peptide containing anamino acid sequence with at least 90% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 8-17, therebyinhibiting the interaction between the CD40 protein and the CD154protein. In one embodiment, the peptide or the functional fragmentthereof binds the CD40 protein. In one embodiment, the CD40 protein ismammalian. In one embodiment, the inhibition is greater than thatexhibited by contacting a peptide or a functional fragment thereofcomprising a sequence set forth in SEQ ID NO: 7.

In one embodiment, the CD40 protein is human. In one embodiment, theCD154 protein is mammalian. In one embodiment, the CD154 protein ishuman. In one embodiment, the CD40 protein is expressed by an immunecell. In one embodiment, the immune cell is selected from the groupconsisting of B-cell, T-cell, neutrophil, monocyte, macrophage,dendritic cell, and platelet. In one embodiment, the contacting inhibitsB-cell activation, T-cell proliferation, T-cell activation, B-cellproliferation, macrophage activation, cytokine production, or acombination thereof. In one embodiment, the CD40 protein is expressed byan endothelial cell, an epithelial cell, a fibroblast cell, a smoothmuscle cell, a B lymphoma cell, a melanoma cell, or a carcinoma cell. Inone embodiment, the contacting comprises administering the peptide or afunctional fragment thereof to a subject. In one embodiment, the subjectis a human.

In one aspect, provided herein is a method of transplantation of a cellor tissue transplant from a mammalian donor in a recipient, the methodcomprising administering an effective amount of the pharmaceuticalcomposition of any one the aspects described above. In another aspect,provided herein is a method of transplantation of a cell, tissue ororgan from a donor to a recipient, the method comprising administeringan effective amount of an anti-CD40 agent; wherein the anti-CD40 agentcomprises a peptide containing an amino acid sequence with at least 85%or at least 90% sequence identity to the sequences set forth in any oneof SEQ ID NOs. 1-6 and 8-17 to the recipient. In another aspect,provided herein is a method of transplantation of a cell, tissue ororgan from a donor to a recipient, the method comprising administeringan effective amount of an anti-CD40 agent; wherein the anti-CD40 agentcomprises a peptide containing an amino acid sequence with at least 90%sequence identity to the sequences set forth in any one of SEQ ID NOs.8-17 to the recipient.

In one embodiment, the peptide is conjugated to apoptotic leukocytes. Inone embodiment, the method further comprises administering apoptoticleukocytes. In one embodiment, the method further comprisesadministering apoptotic leukocytes fixed with a cross-linking agent. Inone embodiment, the method further comprises administering the apoptoticleukocytes and the peptide separately. In one embodiment, the methodfurther comprises administering the apoptotic leukocytes and the peptidesimultaneously. In one embodiment, the method further comprises themethod further comprises transplanting the cell, tissue or organ in therecipient. In one embodiment, the cell or tissue transplant is a kidney,liver, heart, lung, pancreas, islet cell, small bowel, bone marrow,hematopoietic stem cell, embryonic or induced pluripotent stemcell-derived islet beta cell, embryonic or induced pluripotent stemcell-derived islet, embryonic or pluripotent stem-cell derivedhepatocyte or a combination thereof. In one embodiment, the cell ortissue transplant is a xenotransplant. In one embodiment, the cell ortissue transplant is an allotransplant. In one embodiment, theadministering is subcutaneous, intravenous, intradermal,intraperitoneal, oral, intramuscular, intracerebroventricular,intranasal, intracranial, intracelial, intracerebellar, intrathecal,transdermal, pulmonary, or topical administration. In one embodiment,the administering is intravenous administration. In one embodiment, theadministering is performed prior to, during, and after thetransplantation. In one embodiment, the administering is performed atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to thetransplantation. In one embodiment, the administering is performedduring the transplantation. In one embodiment, the administering isperformed at least 1, 2, 3, 4, 5, 6, 7, 10, or more days after thetransplantation. In one embodiment, administering a tolerizing agent tothe recipient. In one embodiment, the tolerizing agent comprisesmammalian leukocytes fixed with a crosslinking agent. In one embodiment,the mammalian leukocytes are from the mammalian donor or from a donorthat is MEW matched to the mammalian donor. In one embodiment, themammalian donor is genetically modified. In one embodiment,administering an immunomodulatory agent to the recipient. In oneembodiment, the immunomodulatory agent is an anti-CD40 agent, anti-CD40Lagent, a B-cell depleting or modulating agent, an mTOR inhibitor, aTNF-alpha inhibitor, a IL-6 inhibitor, alpha 1antitrypsin inhibitor,dehydroxymethylepoxyquinomycin (DHMEQ), a nitrogen mustard alkylatingagent, a complement C3 or C5 inhibitor, IFNγ, an NFκB inhibitor, vitaminD3, siCD40, cobalt protoporphyrin, insulin B9-23, a cluster ofdifferentiation protein, or any combination thereof. In one embodiment,the cell or tissue transplant survives for at least 30 days, 50 days,100 days, 1 year or more in the recipient. In one embodiment, therecipient is a mammal. In one embodiment, the recipient is a human.

In one aspect, provided herein is a method for inducingimmunosuppression in a subject in need thereof, the method comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition in any one of the aspects described above. In one aspect,provided herein is a method of inducing immunosuppression in a subjectin need thereof, the method comprising; administering to the recipientan effective amount of an anti-CD40 agent, wherein the agent is apeptide comprising an amino acid sequence with at least 85% or at least90% sequence identity to the sequences set forth in any one of SEQ IDNOs. 1-6 and 8-17. In one aspect provided herein is a method of inducingimmunosuppression in a subject in need thereof, the method comprising;administering to the recipient an effective amount of an anti-CD40agent, wherein the agent is a peptide comprising an amino acid sequencewith at least 90% sequence identity to the sequences set forth in anyone of SEQ ID NOs. 8-17. In one embodiment, the method comprises furtheradministering apoptotic leukocytes with the anti-CD40 agent. In oneembodiment, the peptide is conjugated to apoptotic leukocytes. In oneembodiment, the apoptotic leukocytes fixed with a cross-linking agent.In one embodiment, the apoptotic leukocytes and the peptides areadministered separately. In one embodiment, the apoptotic leukocytes andthe peptides are administered simultaneously. In one embodiment, themethod further comprises transplanting a cell, tissue or organ in thesubject. In one embodiment, the immune response comprises B-cellactivation, T-cell proliferation, B-cell proliferation, macrophageactivation, cytokine production, or a combination thereof. In oneembodiment, the subject has undergone, is undergoing or will beundergoing an allotransplant. In one embodiment, the subject hasundergone, is undergoing or will be undergoing a xenotransplant. In oneembodiment, the subject is a human. In one embodiment, inhibits B-cellactivation, T-cell proliferation, T-cell activation, B-cellproliferation, macrophage activation, cytokine production, or acombination thereof, thereby inducing immunosuppression. In oneembodiment, the subject is suffering from or is at a risk of developingan inflammatory disease. In one embodiment, the inflammatory disease isan autoimmune disease.

In one aspect, provided herein is a method of inducing immune tolerancein a subject in need thereof, the method comprising administering to thesubject an effective amount of the pharmaceutical composition of any oneof the aspects described above. In another aspect, provided herein is amethod of inducing tolerance to a cell, tissue or organ transplant in arecipient, the method comprising, administering to the recipient aneffective amount of a composition comprising an anti-CD40 agent, whereinthe agent comprises a peptide containing an amino acid sequence with atleast 85% or at least 90% sequence identity to any one of the sequencesset forth in SEQ ID NOs. 1-6 and 8-17. In another aspect, providedherein is a method of inducing tolerance to a cell, tissue or organtransplant in a recipient, the method comprising, administering to therecipient an effective amount of a composition comprising an anti-CD40agent, wherein the anti-CD40 agent comprises a peptide containing anamino acid sequence with at least 90% sequence identity to the sequencesset forth any one of SEQ ID NOs. 8-17.

In one embodiment, the anti-CD40 agent is conjugated to apoptoticleukocytes. In one embodiment, the method further comprisesadministering apoptotic leukocytes. In one embodiment, the methodfurther comprises administering apoptotic leukocytes fixed with across-linking agent. In one embodiment, the method further comprisesadministering the apoptotic leukocytes and the anti-CD40 agentseparately. In one embodiment, the method further comprisesadministering the apoptotic leukocytes and the anti-CD40 agentsimultaneously. In one embodiment, the method further comprisesapoptotic leukocytes conjugated to one or more peptides derived from aMHC class II molecule of the recipient. In one embodiment, the apoptoticleukocytes comprise cells that have been differentiated from stem cellsex vivo, wherein the stem cells are derived from a donor of the cell,tissue, or organ transplant. In one embodiment, the apoptotic leukocytesare MHC matched to the recipient. In one embodiment, the apoptoticleukocytes are conjugated to one or more peptides derived from a MHCclass I molecule of a donor of the cell, tissue or organ transplant. Inone embodiment, the method further comprises transplanting the cell,tissue or organ transplant. In one embodiment, the transplanting isperformed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore days after administering the composition. In one embodiment, themethod further comprises administering at least one booster dose of thecomposition. In one embodiment, the booster dose is administered atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 21, or 24 daysafter the transplanting. In one embodiment, said tolerance is for aperiod of at least one month. In one embodiment, said tolerance is for aperiod of at least 100 days. In one embodiment, said tolerance is for aperiod of at least one year.

In one aspect, provided herein is a method of post-transplant immunetolerizing a subject comprising administering to said subject: ananti-CD40 agent, wherein the agent comprises a peptide comprising anamino acid sequence with at least 85% or at least 90% sequence identityto the sequences set forth in any one of SEQ ID NOs: 1-6 and 8-17. Inanother aspect, provided herein is a method of post-transplant immunetolerizing a subject comprising administering to said subject: ananti-CD40 agent, wherein the agent comprises a peptide comprising anamino acid sequence with at least 90% sequence identity to the sequencesset forth in any one of SEQ ID NOs: 8-17. In another embodiment, theanti-CD40 agent is conjugated to apoptotic leukocytes.

In one embodiment, the anti-CD40 agent is conjugated to apoptoticleukocytes. In one embodiment, the method further comprisesadministering apoptotic leukocytes. In one embodiment, the methodfurther comprises administering apoptotic leukocytes fixed with across-linking agent. In one embodiment, the method further comprisesadministering the apoptotic leukocytes and the anti-CD40 agentseparately. In one embodiment, the method further comprisesadministering the apoptotic leukocytes and the anti-CD40 agentsimultaneously. In one embodiment, the apoptotic leukocytes are furtherconjugated to one or more peptides derived from a MHC class II moleculeof the recipient. In one embodiment, the apoptotic leukocytes comprisecells that have been differentiated from stem cells ex vivo, wherein thestem cells are derived from a donor of the cell, tissue, or organtransplant. In one embodiment, the apoptotic leukocytes are MHC matchedto the recipient. In one embodiment, the apoptotic leukocytes areconjugated to one or more peptides derived from a MHC class I moleculeof a donor of the cell, tissue or organ transplant. In one embodiment,the method further comprises transplanting the cell, tissue or organtransplant. In one embodiment, the method further comprises apoptoticleukocytes isolated from peripheral blood. In one embodiment, the methodfurther comprises apoptotic leukocytes enriched for B cells by positiveor negative selection. In one embodiment, said apoptotic leukocytes areexpanded at least 200 fold relative to a starting population prior tosaid modulating with said carbodiimide crosslinking agent.

In one aspect, provided herein is a method of treating a subjectsuffering from an inflammatory disease, the method comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of any one of the aspects described above. In one aspect,provided herein is a method of treating a subject suffering from or isat a risk of developing a neoplastic disease characterized by aneoplastic cell that expresses CD40, the method comprising administeringto the subject an effective amount of the pharmaceutical composition ofany one of the aspects described above. In one embodiment, theinflammatory disease is an autoimmune disease. In one embodiment, theinflammatory disease is selected from the group consisting of type Idiabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus,erythematosa, chronic obstructive pulmonary disease, atherosclerosis,Crohn's colitis, ulcerative gastritis, primary biliary cirrhosis,Guillain-Barre syndrome, Psoriasis, Graves' disease, Hashimoto'sThyroiditis, Myasthenia Gravis, Vasculitis, acute lung injury, bronchialasthma and acute respiratory distress syndrome.

In one aspect, provided herein is a method of treating a subjectsuffering from or is at a risk of developing a neoplastic diseasecharacterized by a neoplastic cell that expresses CD40, the methodcomprising administering to the subject an effective amount of thepharmaceutical composition of any of the aspects described above. In oneembodiment, the neoplastic disease is a lymphoma, a myeloma or acarcinoma. In one embodiment, the lymphoma is a B cell lymphoma. In oneembodiment, the subject is a mammal. In one embodiment, the subject ishuman.

In one aspect, provided herein is a method of reducing the number ofdonor cells required for a transplantation in a recipient, the methodcomprising administering an effective amount of the pharmaceuticalcomposition of any one of the aspects described above. In oneembodiment, the donor cells are islet cells, stem cell, inducedpluripotent stem cell-derived islet cells or a combination thereof. Inone embodiment, the donor cells are autologous, allogeneic, andxenogeneic.

In one aspect, provided herein is a kit for transplantation of a cell,tissue or organ transplant in a recipient comprising; (a) a firstcontainer comprising a first composition comprising apoptotic leukocytesfixed in a crosslinking agent, an optionally a container comprising acomposition comprising an anti-CD40 agent, wherein the agent is apeptide comprising an amino acid sequence with at least 85% or at least90% sequence identity to the sequences set forth in any one of SEQ IDNOs:1-6 and 8-17. In another aspect, provided herein is a kit fortransplantation of a cell, tissue or organ transplant in a recipientcomprising; (a) a first container comprising a first compositioncomprising apoptotic leukocytes fixed in a crosslinking agent, anoptionally a container comprising a composition comprising an anti-CD40agent, wherein the agent is a peptide comprising an amino acid sequencewith at least 90% sequence identity to the sequences set forth in anyone of SEQ ID NOs:8-17. The kit of any one of claims Error! Referencesource not found.-227, wherein the anti-CD40 agent is conjugated toapoptotic leukocytes. In one embodiment, the apoptotic leukocytes isfurther conjugated to one or more peptides derived from a MHC class IImolecule of the recipient. In one embodiment, the apoptotic leukocytesare MHC matched to the recipient. In one embodiment, the apoptoticleukocytes are further conjugated to one or more peptides derived from aMHC class I molecule of a donor of the cell, tissue or organ transplant.In one embodiment, the kit further comprises the cell, tissue or organtransplant. In one embodiment, the first container and the optionalsecond container is a bottle, a vial, a syringe, or a test tube. In oneembodiment, the first container and the optional second container is amulti-use container

In one aspect, provided herein is a transplant kit comprising apreparatory regimen and a tolerizing regimen: said preparatory regimencomprising an anti-CD40 agent, wherein the agent comprises one or morepeptides comprising an amino acid sequence with at least 85% or at least90% sequence identity to the sequences set forth in any one of SEQ IDNOs: 8-17; said tolerizing regimen comprising: an anti-CD40 agent,wherein the agent comprises one or more peptides comprising an aminoacid sequence with at least 85% or at least 90% sequence identity to thesequences set forth in any one of the sequences set forth in SEQ ID NOs:8-17; wherein said preparatory regimen is administered to a subjectprior to transplantation, and said tolerizing regimen is administeredpost-transplantation to said subject. In another aspect, provided hereinis a transplant kit comprising a preparatory regimen and a tolerizingregimen: said preparatory regimen comprising an anti-CD40 agent, whereinthe agent comprises one or more peptides comprising an amino acidsequence with at least 85% or at least 90% sequence identity to thesequences set forth in any one of SEQ ID NOs: 1-6; said tolerizingregimen comprising: an anti-CD40 agent, wherein the agent comprises oneor more peptides comprising an amino acid sequence with at least 85% orat least 90% sequence identity to the sequences set forth in any one ofthe sequences set forth in SEQ ID NOs: 1-6; wherein said preparatoryregimen is administered to a subject prior to transplantation, and saidtolerizing regimen is administered post-transplantation to said subject

In one embodiment, the transplant kit further comprises the transplantkit further comprises a cell, tissue or organ transplant. In oneembodiment, the anti-CD40 agent is conjugated to apoptotic leukocytes.In one embodiment, the transplant kit further comprises apoptoticleukocytes. In one embodiment, the transplant kit further comprisesapoptotic leukocytes fixed with a cross-linking agent. In oneembodiment, the transplant kit further comprises apoptotic leukocytesconjugated with one or more peptides derived from a MHC class IImolecule of the recipient. In one embodiment, the transplant kit furthercomprises apoptotic leukocytes conjugated one or more peptides derivedfrom a MHC class I molecule of a donor of the cell, tissue or organtransplant. In one embodiment, the transplant kit further comprisesapoptotic leukocytes that have been differentiated from stem cells exvivo, wherein the stem cells are derived from a donor of the cell,tissue, or organ transplant. In one embodiment, the transplant kitfurther comprises apoptotic leukocytes that are MHC matched to therecipient. In one embodiment, the transplant kit further comprisesinstructions for transplanting the cell, tissue or organ transplant. Inone embodiment, the transplant kit further comprises apoptoticleukocytes isolated from peripheral blood. In one embodiment, saidapoptotic leukocytes of said tolerizing regimen are enriched for B cellsby positive or negative selection. In one embodiment, said apoptoticleukocytes of said tolerizing regimen are expanded at least 200 foldrelative to a starting population prior to contacting with saidcrosslinking agent.

In one aspect provided herein is a method of inhibiting immune responseto a therapeutic agent in a subject, the method comprising administeringto the subject the pharmaceutical composition of any one of aspectsabove.

In some embodiments, the therapeutic agent is a biologic. In someembodiments, the biologic is a protein, vaccine, antibody, aptamer,nucleic acids, DNA, RNA, antisense oligonucleotide, virus and bacteria.

In one aspect provided herein is a method of abrogating or reducing anundesired immune response to a therapy in a subject, the methodcomprising administering to the subject the pharmaceutical compositionof any one of aspects above prior to, in conjugation with or after saidtherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the present disclosure are set forth with particularityin the appended claims. A better understanding of the features andadvantages of the present disclosure can be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the disclosure are utilized, and theaccompanying drawings of which:

FIG. 1 shows binding of CD40 targeting peptides to PBMC. Binding of theCD40 targeting peptides was analyzed by incubating 504 of variousfluorochrome tagged CD40 interacting peptides with PBMC from rhesusmonkeys for 30 minutes at 4° C. and analyzed by flow cytometry. Peptides1 and 3 showed high binding affinity.

FIG. 2 demonstrates identified peptides bind specifically to CD40.Binding of the identified peptides to CD40 was analyzed by ELISA.Briefly, plates were coated with CD40 and then incubated with identifiedpeptides and detected using Flag-tagged rhsCD40. Peptide #2, Peptide #5,and Peptide #7 showed similar binding affinity to Peptide #1.

FIGS. 3A-3E shows newly identified peptides demonstrate robustinhibition of T and B cells proliferation. The ability of the CD40targeting peptides to block CD40-CD40L signaling and inhibit T and Bcell proliferation was assessed in a one-way CFSE-MLR. Each of the FIGS.3A-3E show results of CFSE-MLR performed on PBMC obtained from adifferent Rhesus monkey. FACS analysis demonstrates that Peptide #2 andPeptide #3 inhibited T and B cell proliferation significantly betterthan the CD40 targeting peptide identified earlier. The levels of T celland B cell proliferation was 35-46% in all the 5 animals and for sakeclarity was capped at 25% in the figure for better visualization.

FIG. 4 shows CD40 targeting peptides block B cell activation. Theability of the newly identified peptides to block B cell activation byCD40L was analyzed in a peptide blocking study. Briefly, B cells werepre-incubated with newly identified peptides for 30 minutes beforestimulation with rhCD40L. Activation of the B cells was analyzed bymeasuring the expression levels of MHC class II molecules on the cellsurface by flow cytometry. FACS analysis demonstrates that Peptide #2,Peptide #4 and Peptide #5 have a similar or higher ability to block theB cell activation when compared to peptide listed in Peptide #1.

DETAILED DESCRIPTION OF THE DISCLOSURE

The following discussion of the present disclosure has been presentedfor purposes of illustration and description. The following is notintended to limit the invention to the form or forms disclosed herein.Although the description of the present disclosure has includeddescription of one or more embodiments and certain variations andmodifications, other variations and modifications are within the scopeof the present disclosure, e.g., as may be within the skill andknowledge of those in the art, after understanding the presentdisclosure. It is intended to obtain rights which include alternativeembodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Although various features of the disclosure may be described in thecontext of a single embodiment, the features can also be providedseparately or in any suitable combination. Conversely, although thedisclosure may be described herein in the context of separateembodiments for clarity, various aspects and embodiments can beimplemented in a single embodiment.

The practice of some embodiments disclosed herein employ, unlessotherwise indicated, conventional techniques of immunology,biochemistry, chemistry, molecular biology, microbiology, cell biology,genomics and recombinant DNA, which are within the skill of the art. Seefor example Sambrook and Green, Molecular Cloning: A Laboratory Manual,4th Edition (2012); the series Current Protocols in Molecular Biology(F. M. Ausubel, et al. eds.); the series Methods In Enzymology (AcademicPress, Inc.), PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hamesand G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies,A Laboratory Manual, and Culture of Animal Cells: A Manual of BasicTechnique and Specialized Applications, 6th Edition (R. I. Freshney, ed.(2010)).

Definitions

The following definitions supplement those in the art and are directedto the current application and are not to be imputed to any related orunrelated case, e.g., to any commonly owned patent or application.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the presentdisclosure, the preferred materials and methods are described herein.Accordingly, the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. It must be noted that, as used in thespecification, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Furthermore,use of the term “including” as well as other forms, such as “include”,“includes,” and “included,” is not limiting.

The terms “and/or” and “any combination thereof” and their grammaticalequivalents as used herein, can be used interchangeably. These terms canconvey that any combination is specifically contemplated. Solely forillustrative purposes, the following phrases “A, B, and/or C” or “A, B,C, or any combination thereof” can mean “A individually; B individually;C individually; A and B; B and C; A and C; and A, B, and C.”

The term “or” can be used conjunctively or disjunctively, unless thecontext specifically refers to a disjunctive use.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which can depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. It is contemplated that any embodimentdiscussed in this specification can be implemented with respect to anymethod or composition of the invention, and vice versa. Furthermore,compositions of the invention can be used to achieve methods of theinvention.

Reference in the specification to “some embodiments,” “an embodiment,”“one embodiment” or “other embodiments” means that a particular feature,structure, or characteristic described in connection with theembodiments is included in at least some embodiments, but notnecessarily all embodiments, of the inventions.

As used herein, a “subject”, “patient”, “individual” and like terms areused interchangeably and refers to a vertebrate, preferably a mammal,more preferably a primate, still more preferably a human. Mammalsinclude, without limitation, humans, primates, rodents, wild ordomesticated animals, including feral animals, farm animals, sportanimals, and pets. Primates include, for example, chimpanzees,cynomolgous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodentsinclude, for example, mice, rats, woodchucks, ferrets, rabbits andhamsters. Domestic and game animals include, for example, cows, horses,pigs, deer, bison, buffalo, feline species, e.g., domestic cat, andcanine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu,ostrich, and fish, e.g., trout, catfish and salmon. The terms,“individual,” “patient” and “subject” are used interchangeably herein. Asubject can be male or female.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofconditions or disorders associated with CD40 activity. In addition, thecompositions and methods described herein can be used to treatdomesticated animals and/or pets.

A subject can be one who has been previously diagnosed with oridentified as suffering from or under medical supervision for a CD40associated disorder, e.g., inflammatory disease, autoimmune disease,cancer. A subject can be one who is diagnosed and currently beingtreated for, or seeking treatment, monitoring, adjustment ormodification of an existing therapeutic treatment, or is at a risk ofdeveloping a CD40 associated disorder. A subject can be a recipient of atransplant, for example, someone who has undergone, is undergoing, orwill be undergoing transplantation.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agent simultaneously or sequentiallyand in a manner such that their respective effects are additive orsynergistic.

The term “transplant” and its grammatical equivalents as used hereinencompasses any procedure that involves transplantation, implantation,or infusion of cells, tissues, or organs from a donor into a recipient.Non-limiting exemplary types of transplant include autotransplant,autograft, allotransplant, allograft, isotransplant, isograft,xenotransplant, xenograft, and split graft, and domino transplant. In anembodiment, the term “split graft” encompasses any procedure thatinvolves the transplantation of cells, organs, tissues, or evenparticular proteins from a donor is split into more than one recipient.

The term “autotransplantation”, “autotransplant”, “autograft”,“autologous transplantation” or grammatical equivalents as used hereinencompasses any procedure that involves the transplantation of organs,tissues, cells or even particular proteins, or expression products fromone part of the body to another in the same subject. In an embodiment,the subject is a member of a Laurasiatheria super order. In anembodiment, the subject is an ungulate for instance a pig, giraffe,camel, deer or bovine. In an embodiment, the subject is a human ornon-human primate. The autologous tissue (also called autogenous,autogeneic, or autogenic tissue) transplanted by such a procedure iscalled an autograft or autotransplant. Non limiting example can includeautologous transplant of stem cell, induced pluripotent stem cell, cellsderived from stem cells and induced pluripotent cells.

The term “allotransplantation”, “allotransplant”, “allograft” or theirgrammatical equivalents as used herein encompasses any procedure thatinvolves transplantation, implantation, or infusion of cells, tissues,or organs into a recipient, where the recipient and donor are the samespecies. In an embodiment, the recipient and/or donor are a member of aLaurasiatheria super order. In an embodiment, the recipient and/or donorare ungulates for instance pig, giraffe, camel, deer or bovine. In anembodiment, the cells, tissues, or organs described herein aretransplanted into humans or non-human primates. Allotransplantationincludes but is not limited to vascularized allotransplant, partiallyvascularized allotransplant, unvascularized allotransplant,allodressings, allobandages, and allostructures. In some cases, anallotransplant is an isograft or isotransplant in which organs ortissues are transplanted from a donor to a genetically identicalrecipient (such as an identical twin).

The term “xenotransplantation”, “xenotransplant”, “xenograft” or itsgrammatical equivalents as used herein encompasses any procedure thatinvolves transplantation, implantation, or infusion of cells, tissues,or organs into a recipient, where the recipient and donor are differentspecies. In an embodiment, the recipient and/or donor are a member of aLaurasiatheria super order. In an embodiment, the recipient and/or donoris an ungulate, for instance a pig, giraffe, camel, deer or bovine. Inan embodiment, the donor is a pig, and the recipient is a human ornon-human primate. In an embodiment, the cells, tissues, or organsdescribed herein are transplanted into humans or non-human primates.Xenotransplantation includes but is not limited to vascularizedxenotransplant, partially vascularized xenotransplant, unvascularizedxenotransplant, xenodressings, xenobandages, and nanostructures.

The term “transplant rejection” and its grammatical equivalents as usedherein can refer to a process or processes by which an immune responseof an organ transplant recipient mounts a reaction against thetransplanted material (e.g., cells, tissues, and/or organs) sufficientto impair or destroy the function of the transplanted material.

The term “hyperacute rejection” and its grammatical equivalents as usedherein can refer to rejection of a transplanted material or tissueoccurring or beginning within the first 24 hours after transplantation.For example, hyperacute rejection can encompass but is not limited to“acute humoral rejection” and “antibody-mediated rejection”.

“Improving,” “enhancing,” “bettering,” and its grammatical equivalentsas used herein can mean any improvement recognized by one of skill inthe art. For example, improving transplantation can mean lesseninghyperacute rejection, which can encompass a decrease, lessening, ordiminishing of an undesirable effect or symptom.

The term “antibody” as used herein includes IgG (including IgG1, IgG2,IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, andIgY, and is meant to include whole antibodies, including single-chainwhole antibodies, and antigen-binding (Fab) fragments thereof.Antigen-binding antibody fragments include, but are not limited to, Fab,Fab′ and F(ab′)2, Fd (consisting of VH and CH1), single-chain variablefragment (scFv), single-chain antibodies, disulfide-linked variablefragment (dsFv) and fragments comprising either a VL or VH domain. Theantibodies can be from any animal origin. Antigen-binding antibodyfragments, including single-chain antibodies, can comprise the variableregion(s) alone or in combination with the entire or partial of thefollowing: hinge region, CH1, CH2, and CH3 domains. Also included areany combinations of variable region(s) and hinge region, CH1, CH2, andCH3 domains. Antibodies can be monoclonal, polyclonal, chimeric,humanized, and human monoclonal and polyclonal antibodies.

The term “islet”, “islet cells”, “islet equivalent”, “islet-like cells”,“pancreatic islets,” “native islet cells,” “non-native islet cells” andtheir grammatical equivalents as used herein refers to endocrine (e.g.,hormone-producing) cells present in the pancreas of an organism, orcells that mimic one or more function of cells present in the pancreasof an organism. For example, islet cells can comprise different types ofcells, including, but not limited to, pancreatic α cells, pancreatic βcells, pancreatic δ cells, pancreatic F cells, and/or pancreatic εcells. Islet cells can also refer to a group of cells, cell clusters, orthe like, including cells cultured in-vitro. In some embodiments, isletcells are extracted from an islet donor and implanted or transplanted ata predetermined site of an islet recipient for differentiation,expansion, and vascularization to form a therapeutic dose of β-cell massby methods, systems, and/or reagents described herein. In an embodiment,the predetermined site is a renal subcapsular space of the isletrecipient. The islets of Langerhans are the regions of the pancreas thatcontain the endocrine (e.g., hormone-producing) cells (e.g., betacells). In some embodiments, provided herein are neonatal islet cluster(NICC) or neonatal porcine islet (NPI) comprising pancreas lineage cells(e.g., beta-like cells or a cell population comprising beta-like cells)isolated from a donor by methods, systems, and/or reagents describedherein. In some embodiments, NICCs or NPIs are extracted from an isletdonor and implanted or transplanted at a predetermined site of an isletrecipient for differentiation, expansion, and vascularization to form atherapeutic dose of β-cell mass by methods, systems, and/or reagentsdescribed herein. In an embodiment, the predetermined site is a renalsubcapsular space of the islet recipient. In some embodiments, NICCs orNPIs extracted from the donor are implanted or transplanted to therecipient under the cover of transient immunosuppression. In someembodiments, islet cells can be stem cell-derived islet cells, inducedpluripotent stem cell-derived islet cells, transdifferentiated, orsurrogate islet cells. A “donor” is meant to include any mammalianorganism, human or non-human, that can serve as a source of donor tissueor cells for transplantation and/or for inducing donor cell tolerance.Non-human mammals include, but are not limited to, ungulates, such as aneven-toed ungulate (e.g., pigs, peccaries, hippopotamuses, camels,llamas, chevrotains (mouse deer), deer, giraffes, pronghorn, antelopes,goat-antelopes (which include sheep, goats and others), or cattle) or anodd-toed ungulate (e.g., horse, tapirs, and rhinoceroses), a non-humanprimate (e.g., a monkey, or a chimpanzee), a Canidae (e.g., a dog) or acat. A non-human animal can be a member of the Laurasiatheriasuperorder. The donor can be a living donor or a cadaveric donor. Insome cases, the donor is a living donor. In some cases, the donor is acadaveric donor. The cadaveric donor may be, for example, a brain dead,heart beating donor (BDD). The cadaveric donor may be, for example, anon-heart beating donor (NHBD). Whether the donor is a living donor or acadaveric donor (e.g., a BDD or NHBD), the donor can be from any animal,for example, a human or non-human animal. The donor can be in any stageof development, including, but not limited to fetal, perinatal,neonatal, pre-weaning, post-weaning, juvenile, young adult, or adult. Adonor of cells used in the preparation of a tolerizing vaccine orpreparatory regimen can be fully or partially MHC (majorhistocompatibility complex) matched to a transplant donor (e.g., a donorof cells, tissues, or organs used for transplantation). In some cases,the partially matched donor is haploidentical to the transplant donor.In some cases, the partially matched donor shares one or more MHCalleles with a transplant donor. For example, the partially matcheddonor can share one or more of a MHC class I A allele, a MHC class I Ballele, a MHC class II DR allele, a MHC class II DQ allele, or acombination thereof with a transplant donor. The partially matched donorcan share one DR allele with the transplant donors.

A “recipient” can be a human or non-human animal that can receive, isreceiving, or has received a transplant graft, a tolerizing vaccine, apreparatory regimen for transplantation, and/or other compositionsprovided in the present disclosure. A recipient can also be in need of atransplant graft, a tolerizing vaccine, a preparatory regimen fortransplantation, and/or other compositions provided herein. In somecases, the recipient can be a human or non-human animal that canreceive, is receiving, or has received a transplant graft. In somecases, the recipient can be a human or non-human animal that canreceive, is receiving, or has received the presently describedtolerizing vaccine or preparatory regimen for transplantation.

The term “non-human animal” and its grammatical equivalents as usedherein includes all animal species other than humans, includingnon-human mammals, which can be a native animal or a geneticallymodified non-human animal. A non-human mammal includes, an ungulate,such as an even-toed ungulate (e.g., pigs, peccaries, hippopotamuses,camels, llamas, chevrotains (mouse deer), deer, giraffes, pronghorn,antelopes, goat-antelopes (which include sheep, goats and others), orcattle) or an odd-toed ungulate (e.g., horse, tapirs, and rhinoceroses),a non-human primate (e.g., a monkey, or a chimpanzee), a Canidae (e.g.,a dog) or a cat. A non-human animal can be a member of theLaurasiatheria superorder. The Laurasiatheria superorder can include agroup of mammals as described in Waddell et al., Towards Resolving theInterordinal Relationships of Placental Mammals. Systematic Biology 48(1): 1-5 (1999). Members of the Laurasiatheria superorder can includeEulipotyphla (hedgehogs, shrews, and moles), Perissodactyla(rhinoceroses, horses, and tapirs), Carnivora (carnivores),Cetartiodactyla (artiodactyls and cetaceans), Chiroptera (bats), andPholidota (pangolins). A member of Laurasiatheria superorder can be anungulate described herein, e.g., an odd-toed ungulate or even-toedungulate. An ungulate can be a pig. A member can be a member ofCarnivora, such as a cat, or a dog. In some cases, a member of theLaurasiatheria superorder can be a pig.

The term “porcine”, “porcine animal”, “pig” and “swine” and itsgrammatical equivalents as used herein can refer to an animal in thegenus Sus, within the Suidae family of even-toed ungulates. For example,a pig can be a wild pig, a domestic pig, mini pigs, a Sus scrofa pig, aSus scrofa domesticus pig, or inbred pigs.

The term “fetal animal” and its grammatical equivalents can refer to anyunborn offspring of an animal. In some cases, pancreatic cell or tissueare isolated from 6 weeks old embryonic pig for transplantation. Theterm “perinatal animal” and its grammatical equivalents can refer to ananimal immediately before or after birth. For example, a perinatalperiod can start from 20th to 28th week of gestation and ends 1 to 4weeks after birth. The term “neonatal animal” and its grammaticalequivalents can refer to any new born animals. For example, a neonatalanimal can be an animal born within a month. The term “pre-weaningnon-human animal” and its grammatical equivalents can refer to anyanimal before being withdrawn from the mother's milk. The term “juvenileanimal” and its grammatical equivalents can refer to any animal beforebecoming a young adult animal. For example, a juvenile stage of pigs canrefer to any pigs of 2 years of age or younger.

The term “genetically modified”, “genetically engineered”, “transgenic”,“genetic modification” and its grammatical equivalents as used hereinrefer to having one or more alterations of a nucleic acid, e.g., thenucleic acid within an organism's genome. For example, geneticmodification can refer to alterations, additions, and/or deletion ofgenes. A genetically modified cell can also refer to a cell with anadded, deleted and/or altered gene. A genetically modified cell can befrom a genetically modified non-human animal. A genetically engineeredcell from a genetically engineered non-human animal can be a cellisolated from such genetically engineered non-human animal. Agenetically modified cell from a genetically modified non-human animalcan be a cell originated from such genetically modified non-humananimal. A genetically engineered cell or a genetically engineered animalcan comprise a transgene, or other foreign DNA, added or incorporated,or an endogenous gene modified, including, targeted, recombined,interrupted, deleted, disrupted, replaced, suppressed, enhanced, orotherwise altered, to mediate a genotypic or phenotypic effect in atleast one cell of the animal, and typically into at least one germ linecell of the animal.

The term “transgene” and its grammatical equivalents as used herein canrefer to a gene or genetic material that can be transferred into anorganism. For example, a transgene can be a stretch or segment of DNAcontaining a gene that is introduced into an organism. The gene orgenetic material can be from a different species. The gene or geneticmaterial can be synthetic. When a transgene is transferred into anorganism, the organism can then be referred to as a transgenic organism.A transgene can retain its ability to produce RNA or polypeptides (e.g.,proteins) in a transgenic organism. A transgene can comprise apolynucleotide encoding a protein or a fragment (e.g., a functionalfragment) thereof. The polynucleotide of a transgene can be an exogenouspolynucleotide. A fragment (e.g., a functional fragment) of a proteincan comprise at least or at least about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 99% of the amino acid sequence of theprotein. A fragment of a protein can be a functional fragment of theprotein. A functional fragment of a protein can retain part or all ofthe function of the protein. An exogenous polypeptide can encode anexogenous protein or a functional fragment thereof.

The term “exogenous nucleic acid sequence”, “exogenous polynucleotide”and its grammatical equivalents as used herein can refer to a gene orgenetic material that was transferred into a cell or animal thatoriginated outside of the cell or animal. An exogenous nucleic acidsequence can by synthetically produced. An exogenous nucleic acidsequence can be from a different species, or a different member of thesame species. An exogenous nucleic acid sequence can be another copy ofan endogenous nucleic acid sequence.

The term “gene knock-out” or “knock-out” can refer to any geneticmodification that reduces the expression of the gene being “knockedout.” Reduced expression can include no expression. The geneticmodification can include a genomic disruption.

The term “disrupting” and its grammatical equivalents as used herein canrefer to a process of altering a gene, e.g., by deletion, insertion,mutation, rearrangement, or any combination thereof. For example, a genecan be disrupted by knockout. Disrupting a gene can be partiallyreducing or completely suppressing expression (e.g., mRNA and/or proteinexpression) of the gene. Disrupting can also include inhibitorytechnology, such as shRNA, siRNA, microRNA, dominant negative, or anyother means to inhibit functionality or expression of a gene or protein.

The term “gene editing” and its grammatical equivalents as used hereincan refer to genetic engineering in which one or more nucleotides areinserted, replaced, or removed from a genome. For example, gene editingcan be performed using a nuclease (e.g., a natural-existing nuclease oran artificially engineered nuclease).

The term “condition” and its grammatical equivalents as used herein canrefer to a disease, event, or change in health status.

The term “diabetes” and its grammatical equivalents as used herein canrefer to is a disease characterized by high blood sugar levels over aprolonged period. For example, the term “diabetes” and its grammaticalequivalents as used herein can refer to all or any type of diabetes,including, but not limited to, type 1, type 2, type 3c (pancreatogenicdiabetes including cystic fibrosis-related, and surgical, andhemochromatosis-related), gestational diabetes, and monogenic diabetes(HNF1A-MODY, GCK-MODY 2, etc.), and other forms of mitochondrialdiabetes. In some cases, diabetes can be a form of hereditary diabetes.

As used herein, the term “susceptible to” refers to an individual who is“susceptible to” a disease, disorder, and/or condition has not beendiagnosed with the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition may not exhibit symptoms of the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, condition, or event (for example, MuscularDystrophy) may be characterized by one or more of the following: (1) agenetic mutation associated with development of the disease, disorder,and/or condition; (2) a genetic polymorphism associated with developmentof the disease, disorder, and/or condition; (3) increased and/ordecreased expression and/or activity of a protein associated with thedisease, disorder, and/or condition; (4) habits and/or lifestylesassociated with development of the disease, disorder, condition, and/orevent (5) having undergone, planning to undergo, or requiring atransplant. In some embodiments, an individual who is susceptible to adisease, disorder, and/or condition will develop the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, and/or condition will not develop the disease,disorder, and/or condition.

The term “phenotype” and its grammatical equivalents as used herein canrefer to a composite of an organism's observable characteristics ortraits, such as its morphology, development, biochemical orphysiological properties, phenology, behavior, and products of behavior.Depending on the context, the term “phenotype” can sometimes refer to acomposite of a population's observable characteristics or traits.

“Inflammatory disorder” means an immune-mediated inflammatory conditionthat affects humans and is generally characterized by dysregulatedexpression of one or more cytokines. Examples of inflammatory disordersinclude skin inflammatory disorders, inflammatory disorders of thejoints, inflammatory disorders of the cardiovascular system, certainautoimmune diseases, lung and airway inflammatory disorders, intestinalinflammatory disorders. Examples of skin inflammatory disorders includedermatitis, for example atopic dermatitis and contact dermatitis, acnevulgaris, and psoriasis. Examples of inflammatory disorders of thejoints include rheumatoid arthritis. Examples of inflammatory disordersof the cardiovascular system are cardiovascular disease andatherosclerosis. Examples of autoimmune diseases include Type 1diabetes, Graves disease, Guillain-Barre disease, Lupus, Psoriaticarthritis, and Ulcerative colitis. Examples of lung and airwayinflammatory disorders include asthma, cystic fibrosis, COPD, emphysema,and acute respiratory distress syndrome. Examples of intestinalinflammatory disorders include colitis and inflammatory bowel disease.Other inflammatory disorders include cancer, hay fever, periodontitis,allergies, hypersensitivity, ischemia, depression, systemic diseases,post infection inflammation and bronchitis. The peptides andcompositions of the invention may also be employed in thenon-therapeutic treatment of inflammation. Examples of non-therapeutictreatment of inflammation include use to relieve normal,non-pathological, inflammation, for example inflammation in the musclesand joints following exercise

As used herein, the terms “protein”, “peptide” and “polypeptide” areused interchangeably to designate a series of amino acid residuesconnected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, “peptide” and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein”, “peptide” and “polypeptide” are used interchangeablyherein when referring to a gene product and fragments thereof.

The terms “increased”/“increase”, “increasing” or “enhance” are all usedherein to generally mean an increase by a statically significant amount;for the avoidance of doubt, the terms “increased”, “increase”, or“enhance”, mean an increase of at least 10% as compared to a referencelevel, for example an increase of at least about 10%, at least about20%, or at least about 30%, or at least about 40%, or at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90% or up to and including a 100% increase or anyincrease between 10-100% as compared to a reference level, or at leastabout a 2-fold, or at least about a 3-fold, or at least about a 4-fold,or at least about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level. The increase can be, for example, at least 10%, atleast 20%, at least 30%, at least 40% or more, and is preferably to alevel accepted as within the range of normal for an individual without agiven disease.

The terms, “decrease”, “reduce”, “reduction”, “lower” or “lowering,” or“inhibit” are all used herein generally to mean a decrease by astatistically significant amount. For example, “decrease”, “reduce”,“reduction”, or “inhibit” means a decrease by at least 10% as comparedto a reference level, for example a decrease by at least about 20%, orat least about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% decrease (e.g., absentlevel or non-detectable level as compared to a reference level), or anydecrease between 10-100% as compared to a reference level. In thecontext of a marker or symptom, by these terms is meant a statisticallysignificant decrease in such level. The decrease can be, for example, atleast 10%, at least 20%, at least 30%, at least 40% or more, and ispreferably down to a level accepted as within the range of normal for anindividual without a given disease.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography [TLC], gelelectrophoresis and high performance liquid chromatography [HPLC], usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound. In some embodiments, thecompositions of the present disclosure are substantially pure.

By “detectable agent” is meant a compound that is linked to a diagnosticagent to facilitate detection. Such a “detectable agent” may becovalently or non-covalently linked to a diagnostic agent, hi addition,the linkage may be direct or indirect Examples of “detectable agents”include, protein purification tags, cytotoxins, enzymes, paramagneticlabels, enzyme substrates, co-factors, enzymatic inhibitors, dyes,radionuclides, chemiluminescent labels, fluorescent markers, growthinhibitors, cytokines, antibodies, and biotin.

As used herein, biological activity refers to the in vitro, ex vivo, orin vivo activities of a compound or physiological responses that resultupon in vivo administration of a compound, composition or other mixture.Biological activity, thus, encompasses therapeutic effects andpharmaceutical activity of such compounds, compositions and mixtures.

Some numerical values disclosed throughout are referred to as, forexample, “X is at least or at least about 100; or 200 [or any numericalnumber].” This numerical value includes the number itself and all of thefollowing:

-   -   i) X is at least 100;    -   ii) X is at least 200;    -   iii) X is at least about 100; and    -   iv) X is at least about 200.

All these different combinations are contemplated by the numericalvalues disclosed throughout. All disclosed numerical values should beinterpreted in this manner, whether it refers to an administration of atherapeutic agent or referring to days, months, years, weight, dosageamounts, etc., unless otherwise specifically indicated to the contrary.

The ranges disclosed throughout are sometimes referred to as, forexample, “X is administered on or on about day 1 to 2; or 2 to 3 [or anynumerical range].” This range includes the numbers themselves (e.g., theendpoints of the range) and all of the following:

-   -   i) X being administered on between day 1 and day 2;    -   ii) X being administered on between day 2 and day 3;    -   iii) X being administered on between about day 1 and day 2;    -   iv) X being administered on between about day 2 and day 3;    -   v) X being administered on between day 1 and about day 2;    -   vi) X being administered on between day 2 and about day 3;    -   vii) X being administered on between about day 1 and about day        2; and    -   viii) X being administered on between about day 2 and about day        3.

All these different combinations are contemplated by the rangesdisclosed throughout. All disclosed ranges should be interpreted in thismanner, whether it refers to an administration of a therapeutic agent orreferring to days, months, years, weight, dosage amounts, etc., unlessotherwise specifically indicated to the contrary.

Pharmaceutical Compositions

Provided herein are peptides capable of binding CD40 and thereby inhibitactivity of CD40, i.e., interaction of CD40 with CD154. The terms “CD40binding peptide” are used interchangeably with “anti-CD40 peptide”. Thepresent disclosure provides pharmaceutical compositions comprising apeptide which comprise an amino acid sequence selected from Table 1 or aportion thereof or a functional fragment thereof. As it relates to thecomposition and methods of the instant disclosure a peptide can be fulllength peptide selected from Table 1 or can be a portion thereof or afunctional fragment thereof. In some embodiments, a peptide of thecompositions and methods disclosed herein are less than 20 amino acidsin length. In some embodiments, the peptide is 4, 6, 8, 10, 12, 14, or15 amino acids in length. In some embodiments, the peptide comprises anamino acid sequence selected from group consisting of SEQ ID NOs: 1-6and 8-17. The sequences of such peptides are shown in Table 1.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:1. In some embodiments, the peptideof the compositions and methods disclosed herein have an amino acidsequence that is at least 80%, at least 85%, at least 90%, at least 95%,at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 2. Insome embodiments, the peptide of the compositions and methods disclosedherein have an amino acid sequence that is at least 80%, at least 85%,at least 90%, at least 95%, at least 97%, at least 99% or 100% identicalto that of SEQ ID NO:3. In some embodiments, the peptide of thecompositions and methods disclosed herein have an amino acid sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 99% or 100% identical to that of SEQ ID NO:4. In someembodiments, the peptide of the compositions and methods disclosedherein have an amino acid sequence that is at least 80%, at least 85%,at least 90%, at least 95%, at least 97%, at least 99% or 100% identicalto that of SEQ ID NO:5. In some embodiments, the peptide of thecompositions and methods disclosed herein have an amino acid sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 99% or 100% identical to that of SEQ ID NO: 6.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:8.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:9.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:10.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:11.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:12.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:13.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:14.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:15.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:16.

In some embodiments, the peptide of the compositions and methodsdisclosed herein have an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO:17.

In some embodiments, the peptide or functional fragment thereof of thepresent disclosure retain, the ability at a minimum, to bind a CD40protein. In some embodiments, the peptide comprises a portion (e.g., afunctional fragment of the peptide) of the wild type sequence selectedfrom SEQ ID NOs 1-6 and 8-17.

Percent (%) amino acid sequence identity for a given peptide sequencerelative to a reference sequence (e.g., unmodified, full length peptideselected from SEQ ID NOs:1-6 and 8-17 is defined as the percentage ofidentical amino acid residues identified after aligning the twosequences and introducing gaps if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Percent (%) amino acidsequence homology for a given polypeptide sequence relative to areference sequence is defined as the percentage of identical or stronglysimilar amino acid residues identified after aligning the two sequencesand introducing gaps if necessary, to achieve the maximum percenthomology. Non identities of amino acid sequences include conservativesubstitutions, deletions or additions that do not affect the biologicalactivity of the peptide. Strongly similar amino acids can include, forexample, conservative substitutions known in the art. Percent identityand/or homology can be calculated using alignment methods known in theart, for instance alignment of the sequences can be conducted usingpublicly available software such as BLAST, Align, ClustalW2. Thoseskilled in the art can determine the appropriate parameters foralignment, but the default parameters for BLAST are specificallycontemplated.

As used herein, the term “wild type peptide” refers to the native,un-modified peptide comprising amino acid sequence selected from SEQ IDNOs 1-6 and 8-17.

As used herein, a “fragment” or “portion” is one that substantiallyretains at least one biological activity normally associated with thatpeptide. In particular embodiments, the “fragment” or “portion”substantially retains all of the activities possessed by the wild typepeptide. By “substantially retains” biological activity, it is meantthat the fragment or portion retains at least about 50%, 60%, 75%, 85%,90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the wildtype peptide (and can even have a higher level of activity than the wildtype peptide). In some embodiments, a fragment or portion of the proteinor polypeptide described herein is at least 4, 5, 6, 7, 8, 9, 10, 11,12, 13, or 14 or more contiguous amino acids and/or less than about 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 contiguous amino acids,including any combination of the foregoing as long as the lower limit isless than the upper limit and retains at least one biological activityassociated with the wild type peptide.

By “retains” biological activity, it is meant that the peptide retainsat least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, ofthe biological activity of the native peptide (and can even have ahigher level of activity than the native polypeptide). A“non-functional” peptide is one that exhibits essentially no detectablebiological activity normally associated with the peptide (e.g., at most,only an insignificant amount, e.g., less than about 10% or even 5%).

As used herein, a “functional” peptide or fragment is one that retainsat least one biological, activity normally associated with wild typepeptide. Preferably, a “functional” peptide or fragment thereof retainsall of the activities possessed by the native, unmodified or full-lengthwild type peptide. A “non-functional” peptide is one that exhibitsessentially no detectable biological activity normally associated withthe wild type peptide (e.g., at most, only an insignificant amount,e.g., less than about 10% or even 5%). The functional activity can betested by one of ordinary skill in the art by the assays described belowand in the Examples herein.

The compositions and methods of the present disclosure contemplate afunctional derivative, functional fragment, variant, analogue orchemical derivative of a peptide disclosed herein (e.g., a peptideselected from Table 1).

In one embodiment, a peptide useful in the methods and compositionsdescribed herein consists of, consists essentially of, or comprises anamino acid sequence, or is a fragment thereof derived from sequenceselected from SEQ ID NOs: 1-15, provided that the peptide or functionalfragment retains at least one biological activity of corresponding fulllength wild type peptide, the biological activity being selected from ata minimum, to bind CD40, inhibiting interaction of CD40 and CD154 ormodulate immune response mediated by CD40.

The peptides or functional fragment thereof described herein cancomprise conservative amino acid substitutions at one or more amino acidresidues, e.g., at essential or non-essential amino acid residues butwill retain a therapeutically or physiologically relevant activity of apeptide as that term is described herein. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, in a conservative substitution variant, a nonessentialamino acid residue in the polypeptide is preferably replaced withanother amino acid residue from the same side chain family.

In some embodiments, peptide can be a variant of wild type peptide. Theterm “variant” as used herein refers to a peptide or nucleic acid thatis “substantially similar” to a wild-type peptide. A molecule is said tobe “substantially similar” to another molecule if both molecules havesubstantially similar structures (i.e., they are at least 50% similar inamino acid sequence as determined by BLASTp alignment set at defaultparameters) and are substantially similar in at least onetherapeutically or physiologically relevant biological activity. Avariant differs from the naturally occurring polypeptide or nucleic acidby one or more amino acid or nucleic acid deletions, additions,substitutions or side-chain modifications, yet retains one or moretherapeutically relevant, specific functions or desired biologicalactivities of the wild type peptide (e.g., binding CD40).

Amino acid substitutions include alterations in which an amino acid isreplaced with a different naturally-occurring or a non-conventionalamino acid residue. Some substitutions can be classified as“conservative,” in which case an amino acid residue contained in apolypeptide is replaced with another naturally occurring amino acid ofsimilar character either in relation to polarity, side chainfunctionality or size. Substitutions encompassed by variants asdescribed herein can also be “non-conservative,” in which an amino acidresidue which is present in a peptide is substituted with an amino acidhaving different properties (e.g., substituting a charged or hydrophobicamino acid with an uncharged or hydrophilic amino acid), oralternatively, in which a naturally-occurring amino acid is substitutedwith a non-conventional amino acid.

“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that the substitutionof even critical amino acids does not reduce the activity of thepeptide, (i.e. the ability of the peptide to penetrate the blood brainbarrier (BBB)). Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984), incorporated by reference in its entirety.) In some embodiments,individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids can alsobe considered “conservative substitutions” if the change does not reducethe activity of the peptide. Insertions or deletions are typically inthe range of about 1 to 5 amino acids. The choice of conservative aminoacids may be selected based on the location of the amino acid to besubstituted in the peptide, for example if the amino acid is on theexterior of the peptide and expose to solvents, or on the interior andnot exposed to solvents.

The term “insertions” or “deletions” are typically in the range of about1 to 5 amino acids. The variation allowed can be experimentallydetermined by producing the peptide synthetically while systematicallymaking insertions, deletions, or substitutions of nucleotides in thesequence using recombinant DNA techniques.

The term “substitution” when referring to a peptide, refers to a changein an amino acid for a different entity, for example another amino acidor amino-acid moiety. Substitutions can be conservative ornon-conservative substitutions.

In alternative embodiments, one can select the amino acid which willsubstitute an existing amino acid based on the location of the existingamino acid, i.e. its exposure to solvents (i.e. if the amino acid isexposed to solvents or is present on the outer surface of the peptide orpolypeptide as compared to internally localized amino acids not exposedto solvents). Selection of such conservative amino acid substitutionsare well known in the art, for example as disclosed in Dordo et al, J.Mol Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol.119(1986); 205-218 and S. French and B. Robson, J. Mol. Evol.19(1983)171. Accordingly, one can select conservative amino acidsubstitutions suitable for amino acids on the exterior of a protein orpeptide (i.e. amino acids exposed to a solvent), for example, but notlimited to, the following substitutions can be used: substitution of Ywith F, T with S or K, P with A, E with D or Q, N with D or G, R with K,G with N or A, T with S or K, D with N or E, I with L or V, F with Y, Swith T or A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent), for example but notlimited to, one can use the following conservative substitutions: whereY is substituted with F, T with A or S, I with L or V, W with Y, M withL, N with D, G with A, T with A or S, D with N, I with L or V, F with Yor L, S with A or T and A with S, G, T or V. In some embodiments,non-conservative amino acid substitutions are also encompassed withinthe term of variants. The term “derivative” as used herein refers topeptides which have been chemically modified, for example but notlimited to by techniques such as ubiquitination, labeling, pegylation(derivatization with polyethylene glycol), lipidation, glycosylation, oraddition of other molecules. A molecule also a “derivative” of anothermolecule when it contains additional chemical moieties not normally apart of the molecule. Such moieties can improve the molecule'ssolubility, absorption, biological half-life, etc. The moieties canalternatively decrease the toxicity of the molecule, eliminate orattenuate any undesirable side effect of the molecule, etc. Moietiescapable of mediating such effects are disclosed in Remington'sPharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., MackPubl.,Easton, Pa. (1990), incorporated herein, by reference, in its entirety.

A “derivative” is defined as a molecule having the amino acid sequenceof a wild-type peptide (e.g., peptide selected from Table 1) or analogthereof, but additionally having a chemical modification of one or moreof its amino acid side groups, alpha-carbon atoms, terminal amino group,or terminal carboxylic acid group for example by ubiquitination,labeling, pegylation (derivatization with polyethylene glycol) oraddition of other molecules. A chemical modification includes, but isnot limited to, adding chemical moieties, creating new bonds, andremoving chemical moieties. Furthermore, one or more side groups, orterminal groups, may be protected by protective groups known to theordinarily-skilled synthetic chemist. The term “functional” when used inconjunction with “derivative” or “variant” refers to a peptide whichpossesses a biological activity that is substantially similar to abiological activity of the entity or molecule of which it is aderivative or variant. By “substantially similar” in this context ismeant that at least 50% of the relevant or desired biological activityof a corresponding wild-type peptide is retained. In some embodiments,the derivatives retains at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or more, including 100% or even more (i.e., thederivative or variant has improved activity relative to wild-type) ofthe wild type peptide.

In some embodiments a peptide useful in the composition and methods ofthe present disclosure is an analog of a wild type peptide (e.g.,selected from Table 1). An “analog” of a molecule such as a peptiderefers to a molecule similar in function to either the entire moleculeor to a fragment thereof. The term “analog” is also intended to includeallelic species and induced variants. Analogs typically differ from wildtype peptides at one or a few positions, often by virtue of conservativesubstitutions. Analogs typically exhibit at least 80 or 90% sequenceidentity with wild type peptides. Some analogs also include unnaturalamino acids or modifications of N or C terminal amino acids. Examples ofunnatural amino acids are, for example but not limited to; disubstitutedamino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine. Other examples of unnatural aminoacids include, but are not limited to the D-amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 2-aminoisobutyricacid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,naphthalene, L-1-naphthalene, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,b-alanine, fluoro-amino acids, designer amino acids such as b-methylamino acids, Ca-methyl amino acids, Na-methyl amino acids, and aminoacid analogs in general. By “covalently bonded” is meant joined eitherdirectly or indirectly (e.g., through a linker) by a covalent chemicalbond.

The compositions and methods of the instant disclosure also contemplatea peptidomimetic. As used herein, a “peptidomimetic” is a compoundcontaining non-peptidic structural elements that is capable of mimickingthe biological action of a parent peptide. A peptidomimetic may or maynot comprise peptide bonds.

A peptide useful for compositions and methods of the present disclosureshould be of a size sufficient to interact with CD40 protein in such amanner as to inhibit interaction of CD40 with CD154. In someembodiments, a peptide of the present disclosure is less than 20 aminoacids in length. A peptide can be, for example, 4, 6, 13 or 15 aminoacids in length. In one embodiment, the peptide consists of an aminoacid selected from the group consisting of SEQ ID NO:1-6 and 8-17. Thesequences of such peptides are shown below in Table 1. In someembodiments, the peptide comprises at least a portion that is capable ofbinding CD40 and inhibiting interaction of CD40 with CD154. In someembodiments, the peptide comprises at least a portion of peptidecomprising an amino acid sequence selected from SEQ ID NOs: 1-6 and8-17. In some embodiments, the peptide of the present disclosure is asshort as possible, yet at a minimum, retains the biological activity orfunctional activity of binding to CD40. In some embodiments, the peptideis a shortest region of contiguous sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 whichretain at a minimum the biological ability or functional activity ofbinding CD40. Useful peptides can also comprise additional regions ofsequence from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO:15, SEQ ID NO: 16, or SEQ ID NO: 17 which surround the shortest possiblecontiguous sequence capable of binding CD40. In some embodiments, thepeptide or portion thereof bind to CD40 at a site which overlaps thebinding site of CD154. In some embodiments, the peptide or portionthereof bind CD40 at a site which is the binding site of CD154. In someembodiments, the peptide or portion thereof bind the CD40 at a sitewhich is not a site of CD154 binding. The peptides of the presentdisclosure can comprise additional sequences and modifications that donot bind a CD40 protein, so long as the additional sequence do not lowerthe biological activity of the peptide than that of unmodified peptide,for example, biological activity of binding CD40. Non-limiting examplesof additional sequences include sequences coding for B-galactosidase,luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotintags, and fluorescent proteins.

In some embodiments, the composition comprises at least one peptide orportion thereof or functional fragment thereof is selected from Table 1.In some embodiments, the composition can comprise for example more thanone peptides or portion thereof or functional fragment thereof disclosedherein (e.g., a peptide selected from Table 1). In some embodiments, thecompositions disclosed herein can comprise at least 2 peptides, at least3 peptides, at least 4 peptides, at least 5 peptides, at least 6peptides, at least 7 peptides, at least 8 peptides, at least 9 peptides,at least 10 peptides, at least 11 peptides, at least 12 peptides, atleast 13 peptides, at least 14 peptides, at least 15 peptides, orportion thereof (e.g., peptides selected from Table 1). In someembodiments, the composition can comprise all the peptides or portionthereof disclosed herein (e.g., peptides in Table 1).

Biological Activity of Peptides

The term “biological activity,” as used herein, refers to one havingstructural, regulatory, or biochemical functions of an unmodifiedmolecule or any function related to or associated with a metabolic orphysiological process. The biological activity can include an improveddesired activity, or a decreased undesirable activity. For example, anentity demonstrates biological activity when it participates in amolecular interaction with another molecule (e.g., binding CD40), whenit has therapeutic value in alleviating a disease condition, when it hasprophylactic value or therapeutic value in modulating an immuneresponse, when it has diagnostic and/or prognostic value in determiningthe presence of a molecule (e.g., cell expressing CD40).

Binding CD40 Protein

The biological activity and/or biological effect of a peptide disclosedherein include at least in part binding to a CD40 protein. As such, thepeptides or functional fragment thereof disclosed herein are CD40binding peptides or functional fragment thereof. In such a binding, thepeptide associates with CD40 to form a complex. An example of complexformation is association of an antigen with an antibody. The binding ofthe peptides disclosed herein to CD40 can be covalent bindinginteractions between proteins include, for example, disulfide bonds,ester bonds, amide bonds and the like or non-covalent bondinginteractions between proteins include, for example, hydrophobicinteractions, van der Waals interactions, ionic interactions, hydrogenbonding interactions and the like. The binding of peptides disclosedherein with CD40 can be reversible (e.g., non-covalent bindinginteractions) or non-reversible (e.g., covalent bonds). Moreover, areversible interaction can be strong or weak, the strength of theinteraction being determined by the forces (e.g., ionic charges,hydrogen binding, van der Walls interactions, etc.) exerted by eachprotein on the other protein in the complex. Factors affecting thestrength of an interaction between two molecules are known to thoseskilled in the art. One useful measure of the strength of bindingbetween two molecules, such as a peptide and a protein, is thedissociation constant (Kd). The term “dissociation constant,” “kineticoff-rate”, “off-rate”, or “koff” as used interchangeably herein, refersto the value indicating the dissociation rate of a ligand (e.g.,peptides disclosed herein) from its target protein (e.g., CD40) orseparation of the ligand and protein complex (e.g., complex of peptidedisclosed herein with a CD40 protein) over time into free ligand andfree protein. In some embodiments, peptides of the present disclosureare those that bind to a CD40 protein with a Kd of no more than about1×10˜6 M, about 1×10˜7 M, or about 1×10˜8 M. In some embodiments of thecompositions disclosed herein, a peptide has a Kd of less than about1×10˜9 M. In one embodiment, a peptide of the present disclosure bindsto a CD40 protein with a Kd of less than 100 nM, less than 50 nM, lessthan 25 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2nM, or less than 1 nM. Methods of measuring and analyzing bindinginteractions between a peptide and a CD40 protein are known by those ofskill in the art e.g., US20180172683A1. Kd can be determined bymeasurement of the kinetics of complex formation and dissociation, e.g.by the SPR method (Biacore). In one aspect provided herein is a complexcomprising a peptide disclosed herein or a portion thereofnon-covalently bound to a CD40 protein.

In some embodiments, peptide of the present disclosure selectively bindswith a CD40 protein in solution, as determined using an assay such as animmunosorbent assay, or on the surface of a cell. As used herein, theterms selectively, selective, specific, and the like, indicate thepeptide has a greater affinity for a CD40 protein than it does forproteins unrelated to the CD40 protein. More specifically, the termsselectively, selective, specific, and the like indicate that theaffinity of the peptide for CD40 is statistically significantly higherthan its affinity for a negative control (e.g., an unrelated proteinsuch as albumin) as measured using a standard assay (e.g., ELISA).Suitable techniques for assaying the ability of a peptide to selectivelyinteract with a CD40 protein are known to those skilled in the art. Suchassays can be in vitro or in vivo assays. Methods of determiningselective binding of peptides with CD40 are described in Examplesherein. Other non-limiting assays to determine selective bindinginclude, but are not limited to, an enzyme-linked immunoassay, acompetitive enzyme-linked immunoassay, a radioimmunoassay, afluorescence immunoassay, a chemiluminescent assay, a lateral flowassay, a flow-through assay, an agglutination assay, a particulate-basedassay (e.g., using particulates such as, but not limited to, magneticparticles or plastic polymers, such as latex or polystyrene beads), animmunoprecipitation assay, an immunoblot assay (e.g., a western blot), aphosphorescence assay, a flow-through assay, a chromatography assay, apolyacrylamide gel electrophoresis (PAGE)-based assay, a surface plasmonresonance assay, a spectrophotometric assay, a particulate-based assay,an electronic sensory assay and a flow cytometric assay. Methods ofperforming such assays are well known to those skilled in the art. Inone embodiment, an assay can be performed using isolated CD40 protein,of CD40 expressed on a surface of a cell in culture, or it can beperformed in a whole animal. Assays can be designed to give qualitative,quantitative or semi-quantitative results, depending on how they areused and the type of result that is desired.

Human CD40 antigen (CD40) is a peptide of 277 amino acids having amolecular weight of 30,600, and a 19 amino acid secretory signal peptidecomprising predominantly hydrophobic amino acids. CD40 is a member ofthe tumor necrosis factor (TNF)/nerve growth factor (NGF) receptorfamily, which is defined by the presence of cysteine-rich motifs in theextracellular region.

As used herein, “CD40” or “CD40 protein” refers to a cell surfaceglycoprotein that is a member of the tumor necrosis factor receptorfamily. Other names in the art for CD40 include: TNFRSF5, p50, CDW40,CD40 receptor and Bp50. As used herein, the term “CD40 protein”,generally refers to an CD40 polypeptide that is similar or identical insequence to a wild-type CD40. In some embodiments, the term “CD40protein” refers to a CD40 polypeptide having an amino acid sequence thatis at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,at least 99%, or 100%, identical to that of a wild-type CD40 and thatretains the ability, at a minimum, to bind CD154. Accordingly in someembodiments, “CD40” can be full length CD40. In some embodiments, “CD40”can be a functional fragment of a full length CD40, a species homologueand/or functional fragments thereof, an ortholog of CD40 and/orfunctional fragments thereof. The CD40 protein can be a mammalian CD40protein. The CD40 polypeptide can also be a functional isoform of thefull length CD40 or functional fragment thereof.

In some embodiments, “CD40” is a wild-type CD40 of human origin, havingthe following amino acid sequence (SEQ ID NO:18), or a functionalfragment thereof.

(SEQ ID NO: 18) MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

(See GenBank Accession No. AAH12419.1 (sequence includes signalpeptide), which is encoded by nucleic acid sequence GENBANK® Accessionno. BC012419.1. The sequences are incorporated herein by reference intheir entirety).

A “functional fragment” or a “portion thereof” as it relates to CD40refers to fragment of the full length CD40 (e.g. corresponding to SEQ IDNO: 18 of at least 10, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 110, at least 120, at least 130, at least 140, at least 150, atleast 160, at least 170, at least 180, at least 190, at least 200 ormore consecutive amino acids of full length wild-type CD40, that has atleast about 70%, 80%, 90%, 100% or more than 100% of the function ofwild-type CD40 of binding CD154 The functional activity can be tested byone of ordinary skill in the art by the assays described in theexamples.

The polypeptide and coding nucleic acid sequences of CD40 protein ofhuman origin and those of a number of animals are publicly available,e.g., from the NCBI website and are contemplated for use in the methodsand compositions herein. Examples include, but are not limited to, Mouse(GenBank Accession No. AAB08705.1), Rat (GenBank Accession No.AAH97949.1), monkey (Genbank Accession No. AWU67715.1), Porcine (GenBankAccession No. AWU67715.1) Bovine (GenBank Accession No. AAI34766.1).

In some embodiments, the CD40 can be a mammalian homolog of human CD40or a functional fragment thereof. In some embodiments, the CD40polypeptide has an amino acid sequence at least 85%, at least 90%, atleast 95%, at least 97% or at least 99% identical to the amino acidsequence of SEQ ID NO:18 and is capable of binding its cognate ligandCD154. In some embodiments, the CD40 is a functional fragment or aportion of SEQ ID NO: 18 of at least 10, at least 20, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 110, at least 120, at least 130, at least 140consecutive amino acids of SEQ ID NO: 18, that has at least about 50%,60%, 70%, 80%, 90%, 100% or more than 100% of the function of wild typeCD40 of binding its ligand CD154. The functional activity can be testedby one of ordinary skill in the art by the assays described above.

Percent (%) amino acid sequence identity for a given polypeptidesequence relative to a reference sequence is defined as the percentageof identical amino acid residues identified after aligning the twosequences and introducing gaps if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Percent (%) amino acidsequence homology for a given polypeptide sequence relative to areference sequence is defined as the percentage of identical or stronglysimilar amino acid residues identified after aligning the two sequencesand introducing gaps if necessary, to achieve the maximum percenthomology. Non identities of amino acid sequences include conservativesubstitutions, deletions or additions that do not affect the biologicalactivity of CD40. Strongly similar amino acids can include, for example,conservative substitutions known in the art. Percent identity and/orhomology can be calculated using alignment methods known in the art, forinstance alignment of the sequences can be conducted using publiclyavailable software software such as BLAST, Align, ClustalW2. Thoseskilled in the art can determine the appropriate parameters foralignment, but the default parameters for BLAST are specificallycontemplated.

In one embodiment, “CD40 protein” useful in the methods and compositionsdescribed herein consists of, consists essentially of, or comprises anamino acid sequence, or is a portion thereof derived from SEQ ID NO: 18,provided that the polypeptide retains at least one biological activityof full length CD40 of SEQ ID NO: 18, the biological activity beingselected from at a minimum, binding to CD154. The CD40 described hereincan comprise conservative amino acid substitutions at one or more aminoacid residues, e.g., at essential or non-essential amino acid residuesbut will retain a therapeutically or physiologically relevant activityof an inhibitory peptide as that term is described herein. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, in a conservativesubstitution variant, a nonessential amino acid residue in thepolypeptide is preferably replaced with another amino acid residue fromthe same side chain family.

In some embodiments, CD40 can be a variant of wild type CD40. The term“variant” as used herein refers to a polypeptide or nucleic acid that is“substantially similar” to a wild-type CD40. A molecule is said to be“substantially similar” to another molecule if both molecules havesubstantially similar structures (i.e., they are at least 50% similar inamino acid sequence as determined by BLASTp alignment set at defaultparameters) and are substantially similar in at least onetherapeutically or physiologically relevant biological activity. Avariant differs from the naturally occurring polypeptide or nucleic acidby one or more amino acid or nucleic acid deletions, additions,substitutions or side-chain modifications, yet retains one or moretherapeutically relevant, specific functions or desired biologicalactivities of the naturally occurring molecule (e.g., maintainsprimitive HSCs in a quiescent state, enhances hematopoieticreconstitution in vivo).

Amino acid substitutions include alterations in which an amino acid isreplaced with a different naturally-occurring or a non-conventionalamino acid residue. Some substitutions can be classified as“conservative,” in which case an amino acid residue contained in apolypeptide is replaced with another naturally occurring amino acid ofsimilar character either in relation to polarity, side chainfunctionality or size. Substitutions encompassed by variants asdescribed herein can also be “non-conservative,” in which an amino acidresidue which is present in a peptide is substituted with an amino acidhaving different properties (e.g., substituting a charged or hydrophobicamino acid with an uncharged or hydrophilic amino acid), oralternatively, in which a naturally-occurring amino acid is substitutedwith a non-conventional amino acid. Also encompassed within the term“variant,” when used with reference to a polynucleotide or polypeptide,are variations in primary, secondary, or tertiary structure, as comparedto a reference polynucleotide or polypeptide, respectively (e.g., ascompared to a wild-type polynucleotide or polypeptide). Polynucleotidechanges can result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence. Variants can also include insertions, deletions orsubstitutions of amino acids in the peptide sequence. To betherapeutically useful, such variants will retain a therapeutically orphysiologically relevant activity as that term is used herein.

In some embodiments, CD40 can be an agonist of wild-type CD40, an analogor a derivative thereof. In some embodiments, the agonist of wild-typeCD40, an analog or a derivative thereof, retains at least one biologicalactivity of full length CD40 of SEQ ID NO: 18, the biological activitybeing selected from at a minimum, to bind CD154. In some embodiments,the agonist of wild-type CD40, an analog or a derivative thereof,retains at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 100% or more than 100% of the biological activity of fulllength CD40 of SEQ ID NO: 18.

The CD40 polypeptide can be recombinant, purified, isolated, naturallyoccurring or synthetically produced. The term “recombinant” when used inreference to a nucleic acid, protein, cell or a vector indicates thatthe nucleic acid, protein, vector or cell containing them have beenmodified by introduction of a heterologous nucleic acid or protein orthe alteration of a native nucleic acid or a protein, or that the cellis derived from a cell so modified. The term “heterologous” (meaning‘derived from a different organism’) refers to the fact that often thetransferred protein was initially derived from a different cell type ora different species from the recipient. Typically the protein itself isnot transferred, but instead the genetic material coding for the protein(often the complementary DNA or cDNA) is added to the recipient cell.Methods of generating and isolating recombinant polypeptides are knownto those skilled in the art and can be performed using routinetechniques in the field of recombinant genetics and protein expression.For standard recombinant methods, see Sambrook et al, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989);Deutscher, Methods in Enzymology 182:83-9(1990); Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, NY (1982). TheCD40 polypeptide can be immobilized onto a solid surface, e.g., a beador array plate.

In some embodiments, the CD40 protein or a portion thereof is expressedon the surface of a cell. Accordingly, the methods and compositions ofthe present disclosure contemplates a cell expressing CD40 on itssurface. The disclosure is not limiting as it relates to the type ofcell expressing CD40 on its surface. The cell can be an immune cell(e.g., B-cell, T-cell, neutrophil, monocyte, macrophage, dendritic cell,platelet), non-immune cell (e.g., an endothelial cell, epithelial cell,smooth muscle cell, fibroblast), or a cancer cell (e.g., B lymphomacell, carcinoma cell, melanoma cell).

In some embodiments, the CD40 expressing cell is in vitro under cellculture conditions. In other embodiments, the cell expressing CD40protein is in vivo. CD40 may be detected on the surface of a cell by anyone of several means known in the art. For example, an antibody specificfor CD40 may be used in a fluorescence-activated cell sorting techniqueto determine whether cells express CD40 and isolate cells expressingCD40. Other methods of detecting cell surface molecules are also usefulin detecting and isolating cell expressing CD40.

Inhibiting Interaction of CD40 and CD154

As used herein, “CD154” refers to a cell surface glycoprotein that is amember of the tumor necrosis factor ligand family. CD40 ligand is thecognate ligand for CD40. Other names in the art for CD40 ligand include:CD40L, CD40LG, CD 40 ligand, TNFSF5, TRAP, gp39, HIGM1, IGM, IMD3, andT-BAM. An exemplary protein sequence for human CD40L is NP_000065.1,which is encoded by nucleic acid sequence NM_000074.2.

A CD40 ligand “protein, polypeptide or peptide”, or CD154 as usedherein, refers to a proteinaceous CD40 ligand component that hassufficient biological activity to be biologically effective.Accordingly, “CD154” include full-length CD154 proteins and polypeptidesand also CD154 that have been subject to non-native processing orbiological modification. Such modifications include truncations,extensions, active domains or fragments, fusion proteins, mutants withsubstantial or sufficient biological activity, peptidomimetics and thelike. Any form of CD154 can be used methods disclosed herein, includingthose isolated and purified from natural sources. CD154 prepared byrecombinant expression can also be used, i.e., those obtained byexpressing a CD40 ligand nucleic acid in a recombinant host cell andcollecting the expressed CD40 ligand protein. CD40 ligands prepared byautomated peptide synthesis are also included.

CD40 is well known in the art to interact with its cognate ligand CD154.One type of interaction is binding interaction and the said binding orinteraction can result in activation of immune response at least, forexample, by B-cell development, lymphocyte activation and proliferation;modulation of antigen presenting cells function; regulation of activityof dendritic cells, macrophages and B cells; induction of production ofinflammatory cytokines in macrophages and dendritic cells; up-regulatingantigen presentation; up-regulating T cell stimulation; and promotingimmunoglobulin class switching in B cells. The binding can result, forexample, in the inhibition of biological activity mediated by CD40. Inone embodiment, peptides of the present disclosure bind to CD40 in sucha manner as to inhibit the interaction of CD40 with CD154. Accordingly,in some embodiments, the biological activity of the peptides of theinstant disclosure can be inhibiting the interaction of a CD40 proteinwith a CD154 protein. Therefore the peptides or functional fragmentthereof disclosed herein are anti-CD40 peptides or functional fragmentsthereof. The peptide can interact with the CD40 protein in such a mannerthat the strength of the interaction between the CD40 protein and aCD154 protein is increased or decreased.

In some embodiments, the peptides disclosed herein can inhibit theinteraction between CD40 and CD154. As used herein, the term “interact”or “interaction”, and the like mean that two molecules come intosufficient physical proximity such that they cause a biologicaldownstream effect e.g., activation of an immune cell. The interactioncan be, for example, a binding interaction. The interaction can becovalent binding interactions between proteins include, for example,disulfide bonds, ester bonds, amide bonds and the like or non-covalentbonding interactions between proteins include, for example, hydrophobicinteractions, van der Waals interactions, ionic interactions, hydrogenbonding interactions and the like. The interaction can be reversible(e.g., non-covalent binding interactions) or non-reversible (e.g.,covalent bonds). Moreover, a reversible interaction can be strong orweak, the strength of the interaction being determined by the forces(e.g., ionic charges, hydrogen binding, van der Walls interactions,etc.) exerted by each protein on the other protein in the complex.Factors affecting the strength of an interaction between two moleculesare known to those skilled in the art. One useful measure of thestrength of binding between two molecules, such as a peptide and aprotein, is the dissociation constant (Kd). KD can be determined bymeasurement of the kinetics of complex formation and dissociation, e.g.by the SPR method (Biacore). “Inhibit” or “inhibiting,” as used herein,e.g., as in “inhibiting an interaction” between two binding partnerssuch as proteins, refers to a process of lowering or reducing theability of a first protein (e.g., CD40 protein) and a second protein(e.g., CD154 protein) to bind or associate, or disrupting an interactionbetween a first protein and a second protein. “Inhibiting aninteraction” between two proteins may involve disrupting one or morecovalent or non-covalent interactions between the first protein and thesecond protein. Covalent bonding interactions between proteins include,for example, disulfide bonds, ester bonds, amide bonds and the like.Non-covalent bonding interactions between proteins include, for example,hydrophobic interactions, van der Waals interactions, ionicinteractions, hydrogen bonding interactions and the like. As it relatesto the present disclosure, inhibiting an interaction binding of onemolecule (e.g., CD40) with another molecule (e.g., CD154) or theinhibition of stimulation of one cell (e.g., cell expressing a CD40protein on its surface) by another cell (e.g., cell expressing CD154 onits surface) can be through steric hindrance, conformational alterationsor other biochemical mechanism (e.g., through binding of peptides ofinstant disclosure to CD40 protein or cell expressing CD40 protein onits surface).

In some embodiments, the peptides of the present disclosure can inhibitan interaction between a CD40 protein and a CD154 protein by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%), at least 90%>, or at least 95%. Insome embodiments, the peptides disclosed herein can completely inhibitbinding of CD40 to CD154. Complete inhibition of binding between CD40and CD154 means that when a CD40 protein is contacted (treated orbrought into proximity with) a peptide disclosed herein in the presenceof a CD154 protein under conditions that would normally allow theinteraction of CD40 and CD154, no such interaction occurs and activationsignals are not stimulated in the CD40-expressing cell. ConsequentlyCD40/CD154 mediated modulation of immune response does not occur.Methods to measure strength of binding between two proteins, forexample, CD40 and CD154, has been discussed above and are known to thoseskilled in the art.

The term “contacting” as used herein, refers to bringing a disclosedagent (e.g., peptides disclosed herein or a portion thereof) and a cell,a target receptor, or other biological entity (e.g., CD40 protein orcell expressing CD40 on its surface) together in such a manner that thecompound can affect the activity of the target, either directly; i.e.,by interacting with the target itself, or indirectly; i.e., byinteracting with another molecule, co-factor, factor, or protein onwhich the activity of the target is dependent. “Contacting” as usedherein, e.g., as in “contacting a CD40 protein” refers to contactingdirectly or indirectly in vitro, ex vivo, or in vivo (i.e. within asubject as defined herein). In some embodiments, the CD40 protein can beisolated, recombinant, synthesized, or immobilized on a solid surface.In some embodiments, the CD40 protein can be expressed on a surface ofcell (e.g. an immune cell or a cancer cell). The cell expressing CD40can be present in an in vitro cell culture condition or in vivo in asubject. Contacting a CD40 protein can include contacting a cellexpressing said CD40 protein. Contacting a CD40 protein can includeaddition of a peptide or a portion thereof, disclosed herein to a samplecomprising a cell expressing a CD40 protein, or administration to asubject. Contacting encompasses administration to a solution, cell,tissue, mammal, subject, patient, or human. Further, contacting a cellincludes adding a peptide or portion thereof, disclosed herein to a cellculture.

In one aspect, provided herein is a method of inhibiting an interactionbetween a CD40 protein and a CD154 protein comprising contacting theCD40 protein with a peptide disclosed herein.

In some embodiments, peptides disclosed herein can interact with anysite on the CD40 protein. In some embodiments, peptides disclosed hereinbind with the CD40 protein at a site that overlaps with the CD154binding site. In some embodiments, a peptide disclosed herein binds theCD40 protein at the CD154 binding site.

Modulation of Immune Response Mediated by CD40-CD154 Interaction

CD40 is well known to play an important role in activation of immuneresponse. B cells are known to constitutively express the CD40 moleculeon the cells membrane (van Kooten C. et al, J. Leukoc. Biol. 2000).Stimulation of CD40 by CD154-expressing activated T cells, results in Bcells activation, proliferation and isotype switching. Recent studieshave indicated that CD40-CD154 interaction can upregulate costimulatorymolecules, activate antigen presenting cells (APCs) and influence T-cellpriming and T-cell-mediated effector of CD40 activities includemodulating cell survival (e.g., functions as cell survival signal),antibody production, antibody isotype switching, production of cytokines(e.g., IL-2, IL-6, IL-8, IL-12, TNF-α, IL-4, IL-5 and IL-10),metalloproteases (e.g., MMP-I/collagenase and MMP-9/gelatinase B) andestablishment of immune memory. CD40 activities further includemodulating production of proteins involved in cell-cell contact oradhesion (e.g., E-selectin, VCAM-1 and ICAM-1). This interaction canactivate macrophages, natural killer (NK) cells, and endothelial cells.CD40 plays a significant role in immune cell function and signaling,including B-cell and T-cell activation by antigen presenting cells, suchas macrophages and dendritic cells. CD40 activation stimulates antibodyproduction, isotype switching, and establishment of memory. CD40activation stimulates production of cytokines, such as IL-2, IL-6, IL-8,IL-12, TNF-α, IL-4, IL-S and IL-10; and metalloproteases, such asMMP-I/collagenase and MMP-9/gelatinase B. CD40 activation stimulatesproduction of proteins involved in cell-cell contact or adhesion, suchas E-selectin, VCAM-1 and ICAM-1. CD40 recognition on target cellsprovides an activation pathway for NK cell cytotoxic activity. Thus,increasing or decreasing cell survival, antibody production, isotypeswitching, establishment of memory, production of cytokines,metalloproteases or proteins involved in cell-cell contact or adhesion,NK cell cytotoxic activity, B-cell activation, B-cell proliferation,T-cell activation, T-cell proliferation, macrophage activation,migration of immune cell can all be effected or inhibited by contactingan appropriate cell expressing CD40 with a peptide of the presentdisclosure (e.g., peptide selected from Table 1 or a pharmaceuticalcomposition disclosed herein

The term “immune response” encompasses but is not limited to one or bothof the following responses: antibody production (e.g., humoralimmunity), and induction of cell-mediated immunity (e.g., cellularimmunity including helper T cell and/or cytotoxic T cell responses).“Immune response” generally refers to innate and acquired immuneresponses including, but not limited to, both humoral immune responses(mediated by B lymphocytes) and cellular immune responses (mediated by Tlymphocytes). An immune response may be beneficial and lead to immunityagainst infectious pathogens, or an immune response may be pathogenicand lead to autoimmune or hypersensitivity disease. Immune responsesagainst foreign viruses, bacteria, fungi, parasites typically representbeneficial adaptive immune responses. Immune responses againstself-tissues, innocuous foreign objects (e.g., dust mite or pollenallergens, etc.), or tissue transplants represent examples of adversemaladaptive immune responses. The term “modulate,” “modulates” or“modulation” refers to enhancement (e.g., an increase) or inhibition(e.g., a reduction) in the specified activity or suppression of aspecified activity. As it relates to the present disclosure,immunosuppression also refers to inhibition of an immune responsemediated by CD40 activity or one that is mediated by interaction of CD40with CD154

As used herein, “modulation of immune response” can be change in thelevel of an immune cell (e.g., B-cell, T-cell, antigen presenting cell,activated B-cell, activated T-cell, activated macrophage), a change inlevel of immunomodulatory molecules (e.g., inflammatory cytokines,chemokines), or a combination thereof. In some embodiments, modulationof immune response can be suppression of immune response orimmunosuppression. As used herein, the terms level, number, count andconcentration can be used interchangeably. The term “immunomodulatorymolecule” as used herein refers to any molecule which is capable ofeffecting the proliferation or activation of the cells of a subject'simmune system. Such molecules include, without limitation, prostaglandinE2 (PGE2), transforming growth factor-0 (TGF-β), indoleamine2,3-dioxygenase (IDO), nitric oxide, hepatocyte growth factor (HGF),interleukin 6 (IL-6) and interleukin 10 (IL-10).

Modulation of immune response can mean increase or decrease of in thelevel of an immune cell. In some embodiments, the peptides of thepresent disclosure inhibit or decrease the immune response by inhibitingthe level of immune cell (e.g., T-cell, B-cell, antigen presenting cell,activated T-cell, activated B-cell, or activated macrophage), inhibitingthe level of immunomodulatory molecules, or a combination thereof).

Accordingly, in some embodiments the biological activity of peptidesdisclosed herein can be inhibition of CD40 activity such as CD40mediated immune response (e.g., B-cell activation, B-cell proliferation,T-cell activation, T-cell proliferation, macrophage activation,inhibition of inflammatory cytokine production, or a combinationthereof).

It will be appreciated by those skilled in the art that both a cellculture system and the immune system of a subject comprise basal levelsof immune cells and immunomodulatory molecules. The phrases basal leveland normal level can be used interchangeably. As used herein, the basallevel of a type of immune cell, or a immunomodulatory molecule, refersto the average number of that cell type, or immunomodulatory molecule,present in a population of individuals considered healthy (i.e., free ofmetabolic, autoimmune, or infectious disease) or the basal level of atype of immune cell, or an immunomodulatory molecule, refers to theaverage level of that cell type, or immunomodulatory molecule, presentin a population of cells that is not-activated. Those skilled in the artare capable of determining if an immune cell, or a population of suchcells, is activated. For example, the expression of CD69, CD25 and/orCD154 proteins by a T-cell indicates that the cell has been activated.For example, the expression of MHC-class II, B220 and CD3 proteins byB-cell indicates that the B-cell has been activated. For example, theexpression of IL-12, iNOS, Arg-1, or IL-1 proteins by macrophageindicates the macrophage has been activated.

Methods to measure immune cells are well known in the art includingmethods based on identifying expression of specific surface markerproteins e.g., by flow cytometry. Level of immune cell can be measure,for example, by measuring proliferation by 3H-Thymidine Uptake,Bromodeoxyuridine Uptake (BrdU), ATP Luminescence, Fluorescent DyeReduction (carboxyfluorescein succinimidyl ester (CFSE)-like dyes);cytokine measurement, for example, using Multi-Analyte ELISArray Kits,bead-based multiplex assay; measuring surface antigen expression, forexample, by flow cytometry; measuring cell cytotoxicity, for example, byTwo-Label Flow Cytometry, Calcein AM Dye Release, Luciferase TransducedTargets, or Annexin V. Methods to measure T-cell responses and B-cellresponses are well known in the art, for example see Expert Rev.Vaccines 9(6), 595-600 (2010), mBio. 2015 Jul.-Aug.; 6(4).

The reference level or basal level of a cell or molecule can be aspecific amount (e.g., a specific concentration) or it can encompass arange of amounts. Basal levels, or ranges, of immune cells andimmunoregulatory molecules are known to those in the art. For example,in a healthy individual, the normal level of CD4+ T-cells present inhuman blood is 500-1500 cells/ml. Basal levels of cells can also bereported as a percentage of a total cell population.

Immune cell number and function, for example may be monitored by assaysthat detect immune cells by an activity such as cytokine production,proliferation, or cytotoxicity. For example, Lymphoproliferation Assay,which assays the ability of T cells to proliferate in response to anantigen can be used as an indicator of the presence of antigen-specificCD4+ helper T cells. Typically, the specimen of purified T cells orPBMCs is mixed with various dilutions of antigen or antigen in thepresence of stimulator cells (irradiated autologous or HLA matchedantigen-presenting cells). After 72-120 h, [3H] thymidine is added, andDNA synthesis (as a measure of proliferation) is quantified by using agamma counter to measure the amount of radiolabeled thymidineincorporated into the DNA. A stimulation index can be calculated bydividing the number of cpm for the specimen by the number of counts perminute in a control. As it relates to a treatment of an inflammatorydisease, the proliferation assay can be used to compare T-cell responsesbefore and after treatment with peptides of the present disclosure orpharmaceutical compositions disclosed herein. Another example of assaythat can be employed for detection of proliferation of immune cells(e.g., T-cell, B-cells) include use of intracellular fluorescent dye,5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in mixedlymphocyte reaction (CFSE-MLR) and determination of proliferating cellsusing flow cytometry. Another example of an assay that can be employedis detection of secreted cytokines by ELISA and ELISPOT Assays.

Cytokine secretion by immune cells in a subject suffering frominflammatory disease or in response to a transplant) may be detected bymeasuring either bulk cytokine production (by an ELISA) or enumeratingindividual cytokine producing immune cells (by an ELISPOT assay).Generally, in an ELISA assay, PBMC specimens are incubated with antigen(with or without antigen-presenting cells), and after a defined periodof time, the supernatant from the culture is harvested and added tomicrotiter plates coated with antibody for cytokines of interest such asIFN-γ, TNF-α, or IL-2. Antibodies ultimately linked to a detectablelabel or reporter molecule are added, and the plates are washed andread. In a modification of the assay, cytokine secretions can bemeasured in samples (e.g., serum or other body fluids) obtained from asubject using ELISA or ELISPOT before and after treatment with thepeptides disclosed herein or pharmaceutical compositions disclosedherein. Other useful assays include, measurement of detection ofintracellular cytokine assay by flow cytometry, measurement of cytokinemRNA levels by RT-PCR and direct cytotoxicity assays of T-cell (See:Clay et al., 2001). Macrophage activation can be determined, forexample, by measuring levels of chemokines such as IL-8/CXCL8,IP-10/CXCL10, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, and RANTES/CCL5, whichare released as chemoattractants for neutrophils, immature dendriticcells, natural killer cells, and activated T cells. Levels ofpro-inflammatory cytokines are released including IL-1 beta/IL-1F2,IL-6, and TNF-alpha can also be measured by assays well known in theart. Levels of proteolytic enzymes, MMP-1, −2, −7, −9, and −12, whichdegrade Collagen, Elastin, Fibronectin, and other ECM components canalso be measured to determine macrophage activation. Leukocytes areattracted by the macrophage via its release of chemokines includingMDC/CCL22, PARC/CCL18, and TARC/CCL17. Levels of activated B-cell can bedetermined, for example, by measuring antigen specific antibodysecretion or detecting activated B-cell specific surface markers such asCD27, CD19, CD20, CD25, CD30, CD69, CD80, CD86, CD135, by assays such asflow cytometry.

In some embodiments, the peptides disclosed herein can reduce the levelof an immune cell to less than about 50%, less than about 45%, less thanabout 40%, less than about 35%, less than about 30%, less than about27%, or equal to about 25% of the total immune cell population. Methodsof measuring different types of T-cells in the T-cell population areknown to those skilled in the art. In some embodiments, binding of apeptide disclosed herein with a CD40 protein decreases the number ofimmune cells by at least 5%, at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95%. In another embodiment, binding of a peptidedisclosed herein with a CD40 protein decreases the number of immunecells by a factor of at least 10, at least 100, at least 1,000, at least10,000. In some embodiments, the level of immune cells is reduced sothat immune cells comprise no more than about 20%>, about 25%, about30%>, about 35%, or about 40%> of the total immune cell population.

Methods of Preparation of Peptides

The peptides can generally be prepared following techniques known in theart. The peptides can also be prepared using standard geneticengineering techniques known to those skilled in the art. For example,the peptide can be produced enzymatically by inserting nucleic acidencoding the peptide into an expression vector, expressing the DNA, andtranslating the DNA into the peptide in the presence of the requiredamino acids. The peptide is then purified using chromatographic orelectrophoretic techniques, or by means of a carrier protein which canbe fused to, and subsequently cleaved from, the peptide. This can bedone by inserting into the expression vector in phase with the peptideencoding sequence, a nucleic acid sequence encoding the carrier protein.The fusion protein-peptide may be isolated using chromatographic,electrophoretic or immunological techniques (such as binding to a resinvia an antibody to the carrier protein). The peptide can be cleavedusing chemical methodology or enzymatically, as by, for example, usinghydrolases.

Peptides of the disclosure can also be prepared using solution methods,by either stepwise or fragment condensations. An appropriately alphaamino-protected amino acid is coupled to an appropriately alpha carboxylprotected amino acid (such protection may not be required depending onthe coupling method chosen) using diimides, symmetrical or unsymmetricalanhydrides, BOP, or other coupling reagents or techniques known to thoseskilled in the art. These techniques may be either or enzymatic. Thealpha amino and/or alpha carboxyl protecting groups are removed and thenext suitably protected amino acid or block of amino acids are coupledto extend the growing peptide. Various combinations of protecting groupsand of chemical and/or enzymatic techniques and assembly strategies canbe used in each synthesis.

A peptide of the instant disclosure can be synthesized by conventionaltechniques. For example, the peptides can be synthesized by chemicalsynthesis using solid phase peptide synthesis. These methods employeither solid or solution phase synthesis methods (see for example, J. M.Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., PierceChemical Co., Rockford 111. (1984) and G. Barany and R. B. Merrifield,The Peptides: Analysis Synthesis, Biology editors E. Gross and J.Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solidphase synthesis techniques; and M Bodansky, Principles of PeptideSynthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer,Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1, forclassical solution synthesis.)

The peptides can be chemically synthesized by Merrifield-type solidphase peptide synthesis. This method may be routinely performed to yieldpeptides up to about 60-70 residues in length, and may, in some cases,be utilized to make peptides up to about 100 amino acids long. Largerpeptides may also be generated synthetically via fragment condensationor native chemical ligation (Dawson et al., 2000, Ann. Rev. Biochem.69:923-960). An advantage to the utilization of a synthetic peptideroute is the ability to produce large amounts of peptides, even thosethat rarely occur naturally, with relatively high purities, i.e.,purities sufficient for research, diagnostic or therapeutic purposes.Solid phase peptide synthesis is described by Stewart et al. in SolidPhase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company,Rockford, III; and Bodanszky and Bodanszky in The Practice of PeptideSynthesis, 1984, Springer-Verlag, New York. At the outset, a suitablyprotected amino acid residue is attached through its carboxyl group to aderivatized, insoluble polymeric support, such as cross-linkedpolystyrene or polyamide resin. “Suitably protected” refers to thepresence of protecting groups on both the alpha-amino group of the aminoacid, and on any side chain functional groups. Side chain protectinggroups are generally stable to the solvents, reagents and reactionconditions used throughout the synthesis, and are removable underconditions which will not affect the final peptide product. Stepwisesynthesis of the oligopeptide is carried out by the removal of theN-protecting group from the initial amino acid, and coupling thereto ofthe carboxyl end of the next amino acid in the sequence of the desiredpeptide. This amino acid is also suitably protected. The carboxyl of theincoming amino acid can be activated to react with the N-terminus of thesupport-bound amino acid by formation into a reactive group, such asformation into a carbodiimide, a symmetric acid anhydride, or an “activeester” group, such as hydroxybenzotriazole or pentafluorophenyl esters.

Appropriate protective groups usable in such syntheses and theirabbreviations will be found in the above text, as well as in J. F. W.McOmie, Protective Groups in Organic Chemistry, (Plenum Press, New York,1973). The common protective groups used herein are t-butyloxycarbonyl(Boc), fluorenylmethoxycarboyl (FMOC), benzyl (Bzl), tosyl (Tos),o-bromo-phenylmethoxycarbonyl (BrCBZ), phenylmethoxycarbonyl (CBZ),2-chloro-phenylmethoxycarbonyl (2-Cl—CBZ),4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), trityl (Trt), formyl(CHD), and tertiary butyl (t-Bu). N-terminal acetylation on thedeprotected N.sup.α-amino group of peptides synthesized using the Bocstrategy can be accomplished with 10% Ac2 O and 5% DIEA in NMP, followedby washing of the peptide resin with NMP and/or CH2 Cl2.

Examples of solid phase peptide synthesis methods include the BOC methodwhich utilized tert-butyloxcarbonyl as the alpha-amino protecting group,and the FMOC method which utilizes 9-fluorenylmethyloxcarbonyl toprotect the alpha-amino of the amino acid residues, both which methodsare well-known by those of skill in the art. Incorporation of N- and/orC-blocking groups may also be achieved using protocols conventional tosolid phase peptide synthesis methods. For incorporation of C-terminalblocking groups, for example, synthesis of the desired peptide istypically performed using, as solid phase, a supporting resin that hasbeen chemically modified so that cleavage from the resin results in apeptide having the desired C-terminal blocking group. To providepeptides in which the C-terminus bears a primary amino blocking group,for instance, synthesis is performed using a p-methylbenzhydrylamine(MBHA) resin, so that, when peptide synthesis is completed, treatmentwith hydrofluoric acid releases the desired C-terminally amidatedpeptide. Similarly, incorporation of an N-methylamine blocking group atthe C-terminus is achieved using N-methylaminoethyl-derivatized DVB,resin, which upon HF treatment releases a peptide bearing anN-methylamidated C-terminus. Blockage of the C-terminus byesterification can also be achieved using conventional procedures. Thisentails use of resin/blocking group combination that permits release ofside-chain peptide from the resin, to allow for subsequent reaction withthe desired alcohol, to form the ester function. FMOC protecting group,in combination with DVB resin derivatized with methoxyalkoxybenzylalcohol or equivalent linker, can be used for this purpose, withcleavage from the support being effected by TFA in dicholoromethane.Esterification of the suitably activated carboxyl function, e.g. withDCC, can then proceed by addition of the desired alcohol, followed byde-protection and isolation of the esterified peptide product.

In one embodiment, the peptides of the present disclosure aremanufactured by solid phase peptide synthesis using Fmoc chemistry. Incertain embodiments, after synthesis the Fmoc group is deprotected atthe N-terminus, the side chain protection group is deprotected, and thepeptide is cleaved from the resin. In one embodiment, the resin is aCl-resin. In one embodiment, the condensation reaction reagent isDIC+HOBT. In one embodiment deprotection is done using Pip. In certainembodiments, the synthesized peptides are purified by RP-HPLC using asolvent of acetonitrile+deionized with TFA as the buffer. In oneembodiment, the peptides are purified by gradient elution.

The peptides of the disclosure may be prepared by standard chemical orbiological means of peptide synthesis. Biological methods include,without limitation, expression of a nucleic acid encoding a peptide in ahost cell or in an in vitro translation system.

Included in the disclosure are nucleic acid sequences that encode thepeptide of the instant disclosure. In one embodiment, the methods andcompositions herein contemplate use of a nucleic acid fragment encodinga peptide disclosed herein (e.g., peptide selected from Table 1).Accordingly, subclones of a nucleic acid sequence encoding a peptide ofthe disclosure can be produced using conventional molecular geneticmanipulation for subcloning gene fragments, such as described bySambrook et al, Molecular Cloning: A Laboratory Manual, Cold SpringsLaboratory, Cold Springs Harbor, N.Y. (2012), and Ausubel et al. (ed.),Current Protocols in Molecular Biology, John Wiley & Sons (New York,N.Y.) (1999 and preceding editions), each of which is herebyincorporated by reference in its entirety. The subclones then areexpressed in vitro or in vivo in bacterial cells to yield a smallerprotein or polypeptide that can be tested for a particular activity.

Accordingly, in one aspect provided herein is an isolated nucleic acidfragment encoding a peptide comprising an amino acid sequence selectedfrom group consisting of SEQ ID NOs: 1-6 and 8-17 or a portion thereof.Further, the disclosure encompasses an isolated nucleic acid encoding apeptide having substantial homology to a peptide selected from groupconsisting of SEQ ID NOs: 1-6 and 8-17. In some embodiments, theisolated nucleic acid fragment encodes a peptide having a sequence thatis at least 80%, at least 85%, at least 90%, at least 95%, at least 97%,at least 99% or 100% identical to that of SEQ ID NO: 1. In someembodiments, the isolated nucleic acid fragment encodes a peptide havingan amino acid sequence that is at least 80%, at least 85%, at least 90%,at least 95%, at least 97%, at least 99% or 100% identical to that ofSEQ ID NO: 2. In some embodiments, the nucleic acid fragment encodes apeptide having an amino acid sequence that is at least 80%, at least85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%identical to that of SEQ ID NO:3. In some embodiments, the nucleic acidfragment encodes a peptide having an amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 99% or 100% identical to that of SEQ ID NO: 4. In someembodiments, the nucleic acid fragment encodes a peptide having an aminoacid sequence that is at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 99% or 100% identical to that of SEQ ID NO:5. In some embodiments, the isolated nucleic acid fragment encodes apeptide having an amino acid sequence that is at least 80%, at least85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%identical to that of SEQ ID NO: 6. In some embodiments, the isolatednucleic acid fragment encodes a peptide having an amino acid sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 99% or 100% identical to that of SEQ ID NO: 8. In someembodiments, the isolated nucleic acid fragment encodes a peptide havingan amino acid sequence that is at least 80%, at least 85%, at least 90%,at least 95%, at least 97%, at least 99% or 100% identical to that ofSEQ ID NO: 9. In some embodiments, the isolated nucleic acid fragmentencodes a peptide having an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO: 10. In some embodiments, theisolated nucleic acid fragment encodes a peptide having an amino acidsequence that is at least 80%, at least 85%, at least 90%, at least 95%,at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 11.In some embodiments, the isolated nucleic acid fragment encodes apeptide having an amino acid sequence that is at least 80%, at least85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%identical to that of SEQ ID NO: 12. In some embodiments, the isolatednucleic acid fragment encodes a peptide having an amino acid sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 99% or 100% identical to that of SEQ ID NO: 13. In someembodiments, the isolated nucleic acid fragment encodes a peptide havingan amino acid sequence that is at least 80%, at least 85%, at least 90%,at least 95%, at least 97%, at least 99% or 100% identical to that ofSEQ ID NO: 14. In some embodiments, the isolated nucleic acid fragmentencodes a peptide having an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% identical to that of SEQ ID NO: 15. In some embodiments, theisolated nucleic acid fragment encodes a peptide having an amino acidsequence that is at least 80%, at least 85%, at least 90%, at least 95%,at least 97%, at least 99% or 100% identical to that of SEQ ID NO: 16.In some embodiments, the isolated nucleic acid fragment encodes apeptide having an amino acid sequence that is at least 80%, at least85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%identical to that of SEQ ID NO: 17.

As used herein, an “isolated nucleic acid” refers to a nucleic acidsegment or fragment which has been separated from sequences which flankit in a naturally occurring state, i.e., a DNA fragment which has beenremoved from the sequences which are normally adjacent to the fragment,i.e., the sequences adjacent to the fragment in a genome in which itnaturally occurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence. In the context of the present invention, thefollowing abbreviations for the commonly occurring nucleic acid basesare used, “A” refers to adenosine, “C” refers to cytosine, “G” refers toguanosine, “T” refers to thymidine, and “U” refers to uridine.

The present disclosure also encompasses recombinant nucleic acidencoding the peptides described herein. A “recombinant” nucleic acid isone that has been created using genetic engineering techniques.

Biological preparation of a peptide disclosed herein involves expressionof a nucleic acid encoding a desired peptide. An expression cassettecomprising such a coding sequence may be used to produce a desiredpeptide for use in the method of the disclosure. In the context of anexpression vector, the vector can be readily introduced into a hostcell, e.g., mammalian, bacterial, yeast or insect cell by any method inthe art. Coding sequences for a desired peptide of the disclosure can becodon optimized based on the codon usage of the intended host cell inorder to improve expression efficiency as demonstrated herein. Codonusage patterns can be found in the literature (Nakamura et al, 2000, NucAcids Res. 28:292). Representative examples of appropriate hosts includebacterial cells, such as streptococci, staphylococci, E. coli,Streptomyces and Bacillus subtilis cells; fungal cells, such as yeastcells and Aspergillus cells; insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3,BHK, HEK 293 and Bowes melanoma cells; and plant cells.

Numerous vectors are known in the art including, but not limited to,linear polynucleotides, polynucleotides associated with ionic oramphiphilic compounds, plasmids, and viruses. Thus, the term “vector”includes an autonomously replicating plasmid or a virus. The term shouldalso be construed to include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example,polylysine compounds, liposomes, and the like. Examples of viral vectorsinclude, but are not limited to, adenoviral vectors, adeno-associatedvirus vectors, retroviral vectors, and the like. The expression vectorcan be transferred into a host cell by physical, biological or chemicalmeans, discussed in detail elsewhere herein. To ensure that the peptideobtained from either chemical or biological synthetic techniques is thedesired peptide, analysis of the peptide composition can be conducted.Such amino acid composition analysis may be conducted using highresolution mass spectrometry to determine the molecular weight of thepeptide.

Alternatively, or additionally, the amino acid content of the peptidecan be confirmed by hydrolyzing the peptide in aqueous acid, andseparating, identifying and quantifying the components of the mixtureusing HPLC, or an amino acid analyzer. Protein sequenators, whichsequentially degrade the peptide and identify the amino acids in order,can also be used to determine definitely the sequence of the peptide.

The isolated nucleic acid can comprise any type of nucleic acid,including, but not limited to DNA and RNA. For example, in oneembodiment, the composition comprises an isolated DNA molecule,including for example, an isolated cDNA molecule, encoding a peptide ofthe disclosure, or functional fragment thereof. In one embodiment, thecomposition comprises an isolated RNA molecule encoding a peptide of theinvention, or a functional fragment thereof. The isolated nucleic acidscan be synthesized using any method known in the art.

The nucleic acid molecules of the present disclosure can be modified toimprove stability in serum or in growth medium for cell cultures.Modifications can be added to enhance stability, functionality, and/orspecificity and to minimize immunostimulatory properties of the nucleicacid molecule of the disclosure. For example, in order to enhance thestability, the 3′-residues may be stabilized against degradation, e.g.,they may be selected such that they consist of purine nucleotides,particularly adenosine or guanosine nucleotides. Alternatively,substitution of pyrimidine nucleotides by modified analogues, e.g.,substitution of uridine by 2′-deoxythymidine is tolerated and does notaffect function of the molecule.

In one embodiment of the present disclosure the nucleic acid moleculecan contain at least one modified nucleotide analogue. For example, theends may be stabilized by incorporating modified nucleotide analogues.Non-limiting examples of nucleotide analogues include sugar- and/orbackbone-modified ribonucleotides (i.e., include modifications to thephosphate-sugar backbone). For example, the phosphodiester linkages ofnatural RNA may be modified to include at least one of a nitrogen orsulfur heteroatom. In preferred backbone-modified ribonucleotides thephosphoester group connecting to adjacent ribonucleotides is replaced bya modified group, e.g., of phosphothioate group. In preferredsugar-modified ribonucleotides, the 2′ OH-group is replaced by a groupselected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R isCi-C6 alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.

Other examples of modifications are nucleobase-modified ribonucleotides,i.e., ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases can bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

In some instances, the nucleic acid molecule comprises at least one ofthe following chemical modifications: 2′-H, 2′-O-methyl, or 2′-OHmodification of one or more nucleotides. In certain embodiments, anucleic acid molecule of the invention can have enhanced resistance tonucleases. For increased nuclease resistance, a nucleic acid molecule,can include, for example, 2′-modified ribose units and/orphosphorothioate linkages. For example, the 2′ hydroxyl group (OH) canbe modified or replaced with a number of different “oxy” or “deoxy”substituents. For increased nuclease resistance the nucleic acidmolecules of the invention can include 2′-O-methyl, 2′-fluorine,2′-O-methoxyethyl, 2′-O-aminopropyl, 2′-amino, and/or phosphorothioatelinkages. Inclusion of locked nucleic acids (LNA), ethylene nucleicacids (ENA), e.g., 2′-4′-ethylene-bridged nucleic acids, and certainnucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U),G-clamp modifications, can also increase binding affinity to a target.

In one embodiment, the nucleic acid molecule includes a 2′-modifiednucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-0-methyl,2′-0-methoxyethyl (2′-0-MOE), 2′-0-aminopropyl (2′-0-AP),2′-0-dimethylaminoethyl (2′-0-DMAOE), 2′-0-dimethylaminopropyl(2′-O-DMAP), 2′-0-dimethylaminoethyloxyethyl (2′-0-DMAEOE), or2′-0-N-methylacetamido (2′-0-NMA). In one embodiment, the nucleic acidmolecule includes at least one 2′-0-methyl-modified nucleotide, and insome embodiments, all of the nucleotides of the nucleic acid moleculeinclude a 2′-0-methyl modification.

Nucleic acid fragments discussed herein include otherwise unmodified RNAand DNA as well as RNA and DNA that have been modified, e.g., to improveefficacy, and polymers of nucleoside surrogates. Unmodified RNA refersto a molecule in which the components of the nucleic acid, namelysugars, bases, and phosphate moieties, are the same or essentially thesame as that which occur in nature, preferably as occur naturally in thehuman body. The art has referred to rare or unusual, but naturallyoccurring, RNAs as modified RNAs, see, e.g., Limbach et al. {NucleicAcids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often termedmodified RNAs, are typically the result of a post-transcriptionalmodification and are within the term unmodified RNA as used herein.Modified RNA, as used herein, refers to a molecule in which one or moreof the components of the nucleic acid, namely sugars, bases, andphosphate moieties, are different from that which occur in nature,preferably different from that which occurs in the human body. Whilethey are referred to as “modified RNAs” they will of course, because ofthe modification, include molecules that are not, strictly speaking,RNAs. Nucleoside surrogates are molecules in which the ribophosphatebackbone is replaced with a non-ribophosphate construct that allows thebases to be presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone.

Modifications of the nucleic acid of the disclosure can be present atone or more of, a phosphate group, a sugar group, backbone, N-terminus,C-terminus, or nucleobase.

Also provided herein is a vector comprising the isolated nucleic acidfragment of the present disclosure inserted. As used herein, a “vector”or “delivery vector” can be a viral or non-viral vector that is used todeliver a nucleic acid to a cell, tissue or subject. Exemplary vectorsinclude, but are not limited to, adeno-associated virus vectors,adenovirus vectors, lentivirus vectors, paramyxovirus vectors,alphavirus vectors and herpes virus vectors.

A “recombinant” vector or delivery vector refers to a viral or non-viralvector that comprises one or more heterologous nucleotide sequences(i.e., transgenes), e.g., two, three, four, five or more heterologousnucleotide sequences. In an embodiment of the disclosure, therecombinant vectors and delivery vectors of the invention encode afusion polypeptide of NAg and a cytokine such as IFN-β, but can alsoinclude one or more additional heterologous nucleotide sequences, forexample, sequences encoding C- or N-terminal modifications and linkermoieties. As used herein, the term “viral vector” or “viral deliveryvector” can refer to a virus particle that functions as a nucleic aciddelivery vehicle, and which comprises the vector genome packaged withina virion. Alternatively, these terms can be used to refer to the vectorgenome when used as a nucleic acid delivery vehicle in the absence ofthe virion. The art is replete with suitable vectors that are useful inthe present disclosure.

In brief summary, the expression of natural or synthetic nucleic acidsencoding a peptide is typically achieved by operably linking a nucleicacid encoding the peptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors to beused are suitable for replication and, optionally, integration ineukaryotic cells. Typical vectors contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the desired nucleic acid sequence. The vectors ofthe present invention may also be used for nucleic acid immunization andgene therapy, using standard gene delivery protocols. Methods for genedelivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346,5,580,859, 5,589,466, incorporated by reference herein in theirentireties. In another embodiment, the disclosure provides a genetherapy vector.

The isolated nucleic acid of the disclosure can be cloned into a numberof types of vectors. For example, the nucleic acid can be cloned into avector including, but not limited to a plasmid, a phagemid, a phagederivative, an animal virus, and a cosmid. Vectors of particularinterest include expression vectors, replication vectors, probegeneration vectors, and sequencing vectors. Further, the vector may beprovided to a cell in the form of a viral vector. Viral vectortechnology is well known in the art and is described, for example, inSambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York), and in other virology and molecularbiology manuals. Viruses, which are useful as vectors include, but arenot limited to, retroviruses, adenoviruses, adeno-associated viruses,herpes viruses, and lentiviruses. In general, a suitable vector containsan origin of replication functional in at least one organism, a promotersequence, convenient restriction endonuclease sites, and one or moreselectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

For example, vectors derived from retroviruses such as the lentivirusare suitable tools to achieve long-term gene transfer since they allowlong-term, stable integration of a transgene and its propagation indaughter cells. Lentiviral vectors have the added advantage over vectorsderived from onco-retroviruses such as murine leukemia viruses in thatthey can transduce non-proliferating cells, such as hepatocytes. Theyalso have the added advantage of low immunogenicity. In one embodiment,the composition includes a vector derived from an adeno-associated virus(AAV). Adeno-associated viral (AAV) vectors have become powerful genedelivery tools for the treatment of various disorders. AAV vectorspossess a number of features that render them ideally suited for genetherapy, including a lack of pathogenicity, minimal immunogenicity, andthe ability to transduce postmitotic cells in a stable and efficientmanner. Expression of a particular gene contained within an AAV vectorcan be specifically targeted to one or more types of cells by choosingthe appropriate combination of AAV serotype, promoter, and deliverymethod

In certain embodiments, the vector also includes conventional controlelements which are operably linked to a regulatory control sequence in amanner which permits its transcription, translation and/or expression ina cell transfected with the plasmid vector or infected with the virusproduced by the disclosure. As used herein, “operably linked” sequencesinclude both expression control sequences that are contiguous with thegene of interest and expression control sequences that act in trans orat a distance to control the gene of interest. Expression controlsequences include appropriate transcription initiation, termination,promoter and enhancer sequences; efficient RNA processing signals suchas splicing and polyadenylation (poly A) signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance secretion ofthe encoded product. A great number of expression control sequences,including promoters which are native, constitutive, inducible and/ortissue-specific, are known in the art and may be utilized. Additionalpromoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operative ly linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1(EF-1a). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

Enhancer sequences found on a vector also regulates expression of thegene contained therein. Typically, enhancers are bound with proteinfactors to enhance the transcription of a gene. Enhancers may be locatedupstream or downstream of the gene it regulates. Enhancers may also betissue-specific to enhance transcription in a specific cell or tissuetype. In one embodiment, the vector of the present invention comprisesone or more enhancers to boost transcription of the gene present withinthe vector.

In order to assess the expression of the peptide, the expression vectorto be introduced into a cell can also contain either a selectable markergene or a reporter gene or both to facilitate identification andselection of expressing cells from the population of cells sought to betransfected or infected through viral vectors. In other aspects, theselectable marker may be carried on a separate piece of DNA and used ina co-transfection procedure. Both selectable markers and reporter genesmay be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers include, forexample, antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al, 2000 FEBS Letters 479: 79-82). Suitable expression systemsare well known and may be prepared using known techniques or obtainedcommercially. In general, the construct with the minimal 5′ flankingregion showing the highest level of expression of reporter gene isidentified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans. Physical methods for introducing a polynucleotide into a hostcell include calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2012,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection. Biological methodsfor introducing a polynucleotide of interest into a host cell includethe use of DNA and RNA vectors. Viral vectors, and especially retroviralvectors, have become the most widely used method for inserting genesinto mammalian, e.g., human cells. Other viral vectors can be derivedfrom lentivirus, poxviruses, herpes simplex virus I, adenoviruses andadeno-associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). In the casewhere a non-viral delivery system is utilized, an exemplary deliveryvehicle is a liposome. The use of lipid formulations is contemplated forthe introduction of the nucleic acids into a host cell (in vitro, exvivo or in vivo). In another aspect, the nucleic acid may be associatedwith a lipid. The nucleic acid associated with a lipid may beencapsulated in the aqueous interior of a liposome, interspersed withinthe lipid bilayer of a liposome, attached to a liposome via a linkingmolecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the disclosure.

The present disclosure includes a composition comprising a host cellwhich comprises a peptide of the disclosure, a nucleic acid encoding apeptide of the disclosure, or a combination thereof. As used herein, theterm “host cell” comprises prokaryotic cells and eukaryotic cells.Exemplary prokaryotic host cells include E. coli, Bacillus subtilis,etc. Exemplary eukaryotic cells include yeast cells, insect cells,mammal cells, etc. In one embodiment, the cell is genetically modifiedto comprise a peptide and/or nucleic acid of the disclosure. In certainembodiments, genetically modified cell is autologous to a subject beingtreated with the composition of the invention. Alternatively, the cellscan be allogeneic, syngeneic, or xenogeneic with respect to the subject.In certain embodiment, the cell is able to secrete or release theexpressed peptide of the invention into extracellular space in order todeliver the peptide to one or more other cells.

The genetically modified cell may be modified in vivo or ex vivo, usingtechniques standard in the art. Genetic modification of the cell may becarried out using an expression vector or using a naked isolated nucleicacid construct. In one embodiment, the cell is obtained and modified exvivo, using an isolated nucleic acid encoding a peptide. In oneembodiment, the cell is obtained from a subject, genetically modified toexpress the peptide and/or nucleic acid, and is re-administered to thesubject. In certain embodiments, the cell is expanded ex vivo or invitro to produce a population of cells, wherein at least a portion ofthe population is administered to a subject in need. In one embodiment,the cell is genetically modified to stably express the peptide. Inanother embodiment, the cell is genetically modified to transientlyexpress the peptide.

The present disclosure provides a scaffold or substrate compositioncomprising a peptide of the disclosure, an isolated nucleic acid of thedisclosure, a host cell comprising the peptide or isolated nucleic acidfragment of the disclosure, or a combination thereof. For example, inone embodiment, a peptide of the disclosure, an isolated nucleic acid ofthe disclosure, a host cell producing the peptide of the disclosure, ora combination thereof is incorporated within a scaffold. In anotherembodiment, a peptide of the disclosure, an isolated nucleic acid of thedisclosure, a host cell producing the peptide of the disclosure, or acombination thereof is applied to the surface of a scaffold. Thescaffold of the disclosure can be of any type known in the art.Non-limiting examples of such a scaffold includes a, hydrogel,electrospun scaffold, foam, mesh, sheet, patch, and sponge.

Peptide Modifications

The compositions and methods of the present disclosure contemplate useof modified peptides, so long as the modified peptide, retains, thebiological activity of the wild type unmodified peptide e.g., bindingCD40. In some aspects of the disclosure, the peptide is altered. Theterm “altered polypeptide” refers to a peptide that includesalterations, such as deletions, additions, and substitutions (generallyconservative in nature as would be known to a person in the art, such asalanines), to the wild type sequence, as long as the peptide retains thedesired biological activity, i.e., binding CD40, or inhibitinginteraction of CD40 and CD154, and/or modulating immune responsesdirected by interaction of CD40 and CD154. These modifications can bedeliberate, as through site-directed mutagenesis, or can be accidental,such as through mutations of artificial hosts, such as geneticallyengineered bacteria, yeast or mammalian cells, that produce theproteins, or errors due to PCR amplification or other recombinant DNAmethods.

Analogue Formation

In some aspects, a peptide of the instant disclosure is a “modifiedpeptide” comprising non-naturally occurring amino acids. In someaspects, the peptides comprise a combination of naturally occurring andnon-naturally occurring amino acids, and in some embodiments, thepeptides comprise only non-naturally occurring amino acids.

“Modified peptide” may include the incorporation of non-natural aminoacids into the peptides of the disclosure, including syntheticnon-native amino acids, substituted amino acids, or one or more D-aminoacids into the peptides (or other components of the composition, withexception for protease recognition sequences) is desirable in certainsituations. D-amino acid-containing peptides exhibit increased stabilityin vitro or in vivo compared to L-amino acid-containing forms. Thus, theconstruction of peptides incorporating D-amino acids can be particularlyuseful when greater in vivo or intracellular stability is desired orrequired. More specifically, D-peptides are resistant to endogenouspeptidases and proteases, thereby providing better oral trans-epithelialand transdermal delivery of linked drugs and conjugates, improvedbioavailability of membrane-permanent complexes (see below for furtherdiscussion), and prolonged intravascular and interstitial lifetimes whensuch properties are desirable. The use of D-isomer peptides can alsoenhance transdermal and oral trans-epithelial delivery of linked drugsand other cargo molecules. Additionally, D-peptides cannot be processedefficiently for major histocompatibility complex class Il-restrictedpresentation to T helper cells, and are therefore less likely to inducehumoral immune responses in the whole organism. Peptide conjugates cantherefore be constructed using, for example, D-isomer forms of cellpenetrating peptide sequences, L-isomer forms of cleavage sites, andD-isomer forms of therapeutic peptides.

Therefore, in some embodiments the peptides as disclosed comprise L andD amino acids, wherein no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10D-amino acids are included. In certain aspects, the peptides comprisemore than 10 D-amino acids, and in certain aspects all the amino acidsof the peptides are D-amino acids.

Also included are peptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Such variants includethose containing residues other than naturally-occurring L-amino acids,e.g., D-amino acids or non-naturally-occurring synthetic amino acids.The peptides disclosed herein may include unnatural amino acids formedby post-translational modification or by introducing unnatural aminoacids during translation. A variety of approaches are available forintroducing unnatural amino acids during protein translation. By way ofexample, special tRNAs, such as tRNAs which have suppressor properties,suppressor tRNAs, have been used in the process of site-directednon-native amino acid replacement (SNAAR). In SNAAR, a unique codon isrequired on the mRNA and the suppressor tRNA, acting to target anon-native amino acid to a unique site during the protein synthesis(described in WO90/05785). However, the suppressor tRNA must not berecognizable by the aminoacyl tRNA synthetases present in the proteintranslation system. In certain cases, a non-native amino acid can beformed after the tRNA molecule is aminoacylated using chemical reactionswhich specifically modify the native amino acid and do not significantlyalter the functional activity of the aminoacylated tRNA. These reactionsare referred to as post-aminoacylation modifications. For example, theepsilon-amino group of the lysine linked to its cognate tRNA (tRNALYs),could be modified with an amine specific photoaffinity label.

Cyclization

In some embodiments, a peptide of the composition and methods disclosedherein is in a cyclic form. In some embodiments, the cyclic peptidecomprises a linkage between amino acids. Cyclic derivatives of thepeptides disclosed herein are also part of the present disclosure.Cyclization may allow the peptide to assume a more favorableconformation for association with other molecules. Cyclization may beachieved using techniques known in the art. For example, disulfide bondsmay be formed between two appropriately spaced components having freesulfhydryl groups, or an amide bond may be formed between an amino groupof one component and a carboxyl group of another component. Cyclizationmay also be achieved using an azobenzene-containing amino acid asdescribed by Ulysse, L., et al, J. Am. Chem. Soc. 1995, 117, 8466-8467.The components that form the bonds may be side chains of amino acids,non-amino acid components or a combination of the two. In an embodimentof the aspects of the disclosure, cyclic peptides may comprise abeta-turn in the right position. Beta-turns may be introduced into thepeptides of the invention by adding the amino acids Pro-Gly at the rightposition.

It may be desirable to produce a cyclic peptide which is more flexiblethan the cyclic peptides containing peptide bond linkages as describedabove. A more flexible peptide may be prepared by introducing cysteinesat the right and left position of the peptide and forming a disulphidebridge between the two cysteines. The two cysteines are arranged so asnot to deform the beta-sheet and turn. The peptide is more flexible as aresult of the length of the disulfide linkage and the smaller number ofhydrogen bonds in the beta-sheet portion. The relative flexibility of acyclic peptide can be determined by molecular dynamics simulations.

Pharmaceutical Salts:

The peptides of the invention may be converted into pharmaceutical saltsby reacting with inorganic acids such as hydrochloric acid, sulfuricacid, hydrobromic acid, phosphoric acid, etc., or organic acids such asformic acid, acetic acid, propionic acid, glycolic acid, lactic acid,pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid,citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, andtoluenesulfonic acids.

Retro-Inverso Peptide:

In yet a further aspect, the peptides or fragments or derivativesthereof can be “retro-inverso peptides.” A “retro-inverso peptide”refers to a peptide with a reversal of the direction of the peptide bondon at least one position, i.e., a reversal of the amino- andcarboxy-termini with respect to the side chain of the amino acid. Thus,a retro-inverso analogue has reversed termini and reversed direction ofpeptide bonds while approximately maintaining the topology of the sidechains as in the native peptide sequence. The retro-inverso peptide cancontain L-amino acids or D-amino acids, or a mixture of L-amino acidsand D-amino acids, up to all of the amino acids being the D-isomer.Partial retro-inverso peptide analogues are polypeptides in which onlypart of the sequence is reversed and replaced with enantiomeric aminoacid residues. Since the retro-inverted portion of such an analogue hasreversed amino and carboxyl termini, the amino acid residues flankingthe retro-inverted portion are replaced by side-chain-analogousa-substituted geminal-diaminomethanes and malonates, respectively.Retro-inverso forms of cell penetrating peptides have been found to workas efficiently in translocating across a membrane as the natural forms.Synthesis of retro-inverso peptide analogues are described in Bonelli,F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A andViscomi, G. C, J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S.Pat. No. 6,261,569, which are incorporated herein in their entirety byreference. Processes for the solid-phase synthesis of partialretro-inverso peptide analogues have been described (EP 97994-B) whichis also incorporated herein in its entirety by reference.

Modification in the N- or C-Terminus of Peptide Chains:

By Conjugation with Polymers-PEGylation

In certain embodiments, the peptides of the disclosure comprise anN-terminal and/or C-terminal modifications. Such modification can thatin certain instances improve activity. For example, in one embodiment,the peptide of the disclosure comprises a butyric acid at the N-terminusof the peptide. In one embodiment, the peptide is PEGylated at theC-terminus of the peptide. In one embodiment, the carboxy terminus ofthe peptide of the present disclosure is amidated. The presentdisclosure encompasses variants of the peptide analogs, including thosewith terminal modifications, without terminal modifications, or havingdifferent terminal modifications.

For example, in one embodiment, a peptide of the present disclosure canhave, for example, a butyric acid at the N-terminus and is PEGylatedand/or amidated at the C-terminus. However, the present disclosure alsoencompasses analogs and derivatives of peptides disclosed herein,including peptides having different terminal modifications or noterminal modifications. In some embodiments, a peptide or compositionsdisclosed herein can be PEGylated. PEGylation is considered as a goldstandard for chemical modifications of peptides. It involves one or morepolyethylene glycol (PEG) chains linked to a protein, peptide ornon-peptide molecule. PEGylation is the term given to conjugation withmonomethoxy poly (ethyleneglycol) or mPEG. PEG polymer, a polyethercompound, or a polymer of ethylene oxide is highly soluble in water.

The peptides of the disclosure may further be conjugated to non-aminoacid moieties that are useful in their therapeutic application. Inparticular, moieties that improve the stability, biological half-life,water solubility, and/or immunologic characteristics of the peptide areuseful. A non-limiting example of such a moiety is polyethylene glycol(PEG).

For maximum benefits, a stable bond is formed between PEG andpolypeptide. To prepare an active PEG derivative with a functionalgroup—such as active ester, active carbonate, tresylate or aldehydesuitable for coupling to a given peptide. Preferred positions forPEGylation are the N-terminal amino group of the polypeptide backboneand the ε-amino group in the side chain of the amino acid residuelysine. Being highly labile, mild chemical reaction conditions are usedby proteins and peptides for the conjugation at the hydrophobic sites.

Polystyrene-Co-Maleic Acid Anhydride) or SMA:

It is a synthetic polymer, made up of styrene and maleic anhydridepolymer, with molecular weight of 1.5 kDa, which on conjugation withproteins and peptide increases systemic circulation time and eliminationhalf life of the conjugated peptide to natural long-circulating bloodplasma components such as serum albumin or lipoproteins.

Polysialation:

It involves the conjugation of peptides and proteins to the naturallyoccurring, biodegradable α-(2→8) linked polysialic acid3. Polysialicacid is highly hydrophilic in nature which increases the systemiccirculation. It has the advantage of being biodegradable and itscatabolic products are not toxic (e.g. NeuNAc). It markedly reducesproteolysis, prolongs the half life in systemic circulation (up to 40 h)and retains the activity of protein and peptide in-vivo. It also reducesimmunogenicity and antigenicity. Polysialic acids are linear polymers ofN-acetylneuraminic acid (sialic acid) abundantly present on the surfaceof cells and many proteins. Polysialylation has been tested and are usedin therapeutics for the treatment of various diseases. It includespreservation of stability and function, optimal pharmacokinetics andpharmacodynamics, and

Covalent attachment of biologically active compounds to water-solublepolymers is one method for alteration and control of biodistribution,pharmacokinetics, and often, toxicity for these compounds (Duncan et al,1984, Adv. Polym. Sci. 57:53-101). Many water-soluble polymers have beenused to achieve these effects, such as poly(sialic acid), dextran,poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA),poly(N-vinylpyrrolidone) (PVP), poly(vinyl alcohol) (PVA), poly(ethyleneglycol-co-propylene glycol), poly(N-acryloyl morpholine (PAcM), andpoly(ethylene glycol) (PEG) (Powell, 1980, Polyethylene glycol. In R. L.Davidson (Ed.) Handbook of Water Soluble Gums and Resins. McGraw-Hill,New York, chapter 18). PEG possess an ideal set of properties: very lowtoxicity (Pang, 1993, J. Am. Coll. Toxicol. 12: 429-456) excellentsolubility in aqueous solution (Powell, supra), low immunogenicity andantigenicity (Dreborg et al, 1990, Crit. Rev. Ther. Drug Carrier Syst.6: 315-365). PEG-conjugated or “PEGylated” protein therapeutics,containing single or multiple chains of polyethylene glycol on theprotein, have been described in the scientific literature (Clark et al.,1996, J. Biol. Chem. 271: 21969-21977; Hershfield, 1997, Biochemistryand immunology of poly(ethylene glycol)-modified adenosine deaminase(PEG-ADA). In J. M. Harris and S. Zalipsky (Eds) Poly(ethylene glycol):Chemistry and Biological Applications. American Chemical Society,Washington, D.C., p 145-154; Olson et al, 1997, Preparation andcharacterization of poly(ethylene glycosylated human growth hormoneantagonist. In J. M. Harris and S. Zalipsky (Eds) Poly(ethylene glycol):Chemistry and Biological Applications. American Chemical Society,Washington, D.C., p 170-181).

Post Translation Modification

In some aspects of all the embodiments of the disclosure, the peptidesor modified peptides further comprise co-translational andpost-translational (C-terminal peptide cleavage) modifications, such as,for example, disulfide-bond formation, glycosylation, acetylation,phosphorylation, proteolytic cleavage (e.g., cleavage by furins ormetalloproteases), and the like. The main post-translationalmodifications associated with peptides are: Acetylation, acylation,ADP-ribosylation, Amidation, γ-Carboxylation and β-hydroxylation,Disulfide bond formation, Glycosylation, Phosphorylation, Proteolyticprocessing, Sulfation, Methylation, Acyl Lipidation. A peptidemodification includes phosphorylation (e.g., on a Tyr, Ser or Thrresidue), N-terminal acetylation, C-terminal amidation, C-terminalhydrazide, C-terminal methyl ester, fatty acid attachment, sulfonation(tyrosine), N-terminal dansylation, N-terminal succinylation,tripalmitoyl-S-Glyceryl Cysteine (PAMb 3 Cys-OH) as well as famesylationof a Cys residue. Systematic chemical modification of a peptide can, forexample, be performed in the process of peptide optimalization. Forexample, post-translational modifications that fall within the scope ofthe present disclosure include signal peptide cleavage, glycosylation,acetylation, isoprenylation, proteolysis, myristoylation, proteinfolding and proteolytic processing, etc. Some modifications orprocessing events require introduction of additional biologicalmachinery. For example, processing events, such as signal peptidecleavage and core glycosylation, are examined by adding caninemicrosomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489)to a standard translation reaction. Modifications (which do not normallyalter primary sequence) include in vivo, or in vitro chemicalderivatization of polypeptides, e.g., acetylation, or carboxylation.Also included are modifications of glycosylation, e.g., those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps; e.g., byexposing the polypeptide to enzymes which affect glycosylation, e.g.,mammalian glycosylating or deglycosylating enzymes. Also embraced aresequences which have phosphorylated amino acid residues, e.g.,phosphotyrosine, phosphoserine, or phosphothreonine.

Site Specific Modifications

The peptides of the disclosure can be conjugated with other molecules,such as peptide tags or proteins, to prepare fusion proteins. This maybe accomplished, for example, by the synthesis of N-terminal orC-terminal fusion proteins provided that the resulting fusion proteinretains the functionality of the peptide of the invention. The term“fusion protein” as used herein refers to a recombinant protein of twoor more proteins. Fusion proteins can be produced, for example, by anucleic acid sequence encoding one protein is joined to the nucleic acidencoding another protein such that they constitute a single open-readingframe that can be translated in the cells into a single polypeptideharboring all the intended proteins. The order of arrangement of theproteins can vary. Fusion proteins can include an epitope tag or ahalf-life extender. Epitope tags include biotin, FLAG tag, c-myc,hemaglutinin, His6, digoxigenin, FITC, Cy3, Cy5, green fluorescentprotein, V5 epitope tags, GST, β-galactosidase, AU1, AUS, and avidin.Half-life extenders include Fc domain and serum albumin. Accordingly, insome embodiments, the peptides disclosed herein can be conjugated to apeptide tag. Other modifications can be made, for example, Fusion of apeptide to a solubilising protein fusion partner such a maltose-bindingprotein (MBP), glutathione S-transferase (GST), green fluorescentprotein (GFP). In some embodiments, a peptide of the present disclosurefurther comprises a peptide tag. Addition of short solubilityenhancement peptide tags containing 5-10 positively charged amino acidsto N- or C-terminus of proteins and peptides. Such peptide tags aresmall relative to large proteins but are in significant fraction forsmaller proteins. Tolbert et al used a betaine moiety, a small molecule,containing a positively charged quaternary ammonium group (MW-100) isintroduced as a small solubility enhancement tag onto the N-terminus ofpolypeptides by chemical ligation or expressed protein ligation. Themodified forms of polypeptides are more water soluble. Modification withsmall ubiquitin related modifier (SUMO) has the ability to enhanceprotein expression and solubility.

In some embodiments, a peptide of the present disclosure can beconjugated to a detectable agent. The term “detectable agent” as usedherein that is conjugated directly or indirectly to a probe (e.g.,peptide) to generate a “labeled” probe. The label may be detectable byitself (e.g. radioisotope labels, fluorescent agent or chemiluminescentagent) or, in the case of an enzymatic label, may catalyze chemicalalteration of a substrate compound or composition that is detectable(e.g., avidin-biotin). In some instances, primers can be labeled todetect a PCR product.

In some embodiments, the peptides of the compositions and methodsdisclosed herein is modified by linkage to a carrier peptide. The term“conjugated” are used to refer to any method known in the art forfunctionally connecting moieties (such as detectable agent, carrierpeptide), including, without limitation, recombinant fusion, covalentbonding, disulfide bonding, ionic bonding, hydrogen bonding, andelectrostatic bonding. The term “carrier peptide” as used hereinindicates peptides that transport a specific substance or group ofsubstances (e.g., peptides of the present disclosure) throughintracellular compartments or in extracellular fluids (e.g. in theblood) or else across the cell membrane. Exemplary carrier proteinscomprise subunit B of cholera toxin, Avidin, BTG protein, Bovine Gglobulin, Bovine Immunoglobulin G, Bovine Thyroglobulin, Bovine SerumAlbumin (BSA), Conalbumin, Edestein, Exoprotein A from Pseudomonasaeruginosa, HC (Hemocyanin from crab Paralithodes camtschatica), HelixPromatia Haemocyanin (HPH), Human Serum Albumin (HSA), KTI (Kunitstrypsin inhibitor from soybeans), Keyhole Limpet Heamocyanin (KLH), LPH(Haemocyanin from Limulus polyphemus) Ovalbumin, Pam3Cys-Th, Polylysine,porcine Thyroglobulin (PTG), Purified Protein Derivative (PPD), RabbitSerum Albumin (RSA), Soybeab Trypsin Inhibitor (STI) (Sunflower Globulin(SFG) and additional molecules identifiable by a skilled person.Additional carriers comprise molecule having immunogenic activitiesincluding cytokines such as IL-10, IL12, IL-4 IL-16 and TransformingGrowth Factor Beta (TGFP).

Accordingly, in some embodiments a peptide of the compositions andmethods disclosed herein is fused to a carrier peptide. In someembodiments, attachment of the carrier is performed at the C-terminus orN-terminus of the fragment. In an embodiment the fusion protein (i.e.,peptides of the present disclosure and a carrier peptide) can beprovided as a single polypeptide through recombinant DNA technology andrelated processes, such as cloning, chimeric constructs, PolymeraseChain Reaction and additional procedures identifiable by a skilledperson. In some embodiments, attachment can be performed throughchemical linkage of the fragment to the carrier using methods alsoidentifiable by a skilled person.

Non-limiting examples of such transportable peptides, or vectors,suitable for coupling to the pharmaceutical agent include transferrin,insulin-like growth factors I and II, basic albumin and prolactin. Theconjugation can be carried out using bifunctional reagents which arecapable of reacting with each of the polypeptides and forming a bridgebetween the two. The preferred method of conjugation involvespolypeptide thiolation, wherein the two polypeptides are treated with areagent such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) toform a disulfide bridge between the two polypeptides. Other knownconjugation agents can be used, so long as they provide linkage of thetwo polypeptides (i.e. therapeutic polypeptide drug and thetransportable peptide) together without denaturing them. Preferably, thelinkage can be easily broken once the chimeric polypeptide has enteredthe region of interest. In some embodiments, the peptides of the presentdisclosure can be linked to short cell penetrating peptides, which havethe ability to cross cell membrane bilayers. Non limiting examples ofsuch peptides include TAT (HIV-1 transactivating transcriptor) SynB3,Tat, transportan.

Variants of suitable peptides of the invention can also be expressed.Variants may be made by, for example, the deletion, addition, oralteration of amino acids that have either (i) minimal influence oncertain properties, secondary structure, and hydropathic nature of thepolypeptide or (ii) substantial effect on one or more properties of thepeptide mimetics of the invention. Variants may also include, forexample, a peptide conjugated to a linker or other sequence for ease ofsynthesis, purification, identification, or therapeutic use (i.e.,delivery) of the peptide. The variants of the peptides according to thepresent invention may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code,(ii) one in which there are one or more modified amino acid residues,e.g., residues that are modified by the attachment of substituentgroups, (iii) one in which the peptide is an alternative splice variantof the peptide of the present invention, (iv) fragments of the peptidesand/or (v) one in which the peptide is fused with another peptide, suchas a leader or secretory sequence or a sequence which is employed forpurification (for example, His-tag) or for detection (for example, Sv5epitope tag). The fragments include peptides generated via proteolyticcleavage (including multi-site proteolysis) of an original sequence.Variants may be post-translationally, or chemically modified. Suchvariants are deemed to be within the scope of those skilled in the artfrom the teaching herein.

Conjugation with Fatty Acids (Lipidization)

Delivery to and uptake of peptides is favored by low molecular weight,lack of ionization at physiological pH and lipophilicity. Therefore onepossible strategy to improve targeting of the compositions of thepresent disclosure is to modify the peptides to increase itslipophilicity. Accordingly, in some embodiments, the peptides disclosedherein can comprise a lipophilic molecular group. As used herein theterm “lipophilic molecular group” refers to a lipid moiety, such as afatty acid, glyceride or phospholipid which when coupled to atherapeutic molecule, increases its lipophilicity. The lipophilicmolecular group can be attached to the therapeutic molecule through anester bond. Examples of such modifications include, among others,esterification, or amidation of the hydroxy-, amino-, or carboxylicacid-groups of the polypeptide. Lipophilic molecular groups can compriselipid moieties such as fatty acid, glyceride or phospholipids.

Lipidization can involve acylation of the ε-amino group of lysineresidues or acylation of N-terminal α-amino groups. The presence of alipid group in peptides changes their secondary structures,hydrophobicity and self-assembling propensities but retains its abilityto bind to target receptors.

Reversible Aqueous lipidization technology (REAL) This is a new methodfor developing fatty acids-peptide conjugates which is carried out inaqueous solution and can regenerate the original active peptides intissues or the blood and thus called REAL Technology. Lipidization canbe carried out at cysteine residues following cleavage ofintra-disulfide bonds with a reducing agent. This type of lipidizationis typically reversible. Methods of REAL are described in, for example,Shen et al. In some embodiments, a modification combining site specificmodification and lipidization technique is carried out, e.g., Chang et.al. L Pharm Sci 2002.

Hydrophobic Ion Pairing

In some embodiments, modification of a peptide disclosed herein is amodification by adding an opposite charged surfactant that binds topeptide to obtain neutral hydrophobicity entity. Solubility of ioniccompounds is high due to ease of solvation of the counter ions withionic detergents. Positively charged peptides and negatively chargedsurfactants should be employed, since cationic surfactants might havetoxic side effects. Replacement of counter ions decreases the aqueoussolubility and increases the lipophilicity. Thus, it changes thepartition coefficient by orders of magnitude. HIP method is inexpensiveand reversible. The process involves replacement of counter ions (e.g.chloride, acetate, nitrate) with an ionic detergent of similar charge.For many proteins, dissolution in organic solvents occurs with retentionof native-like structure and maintenance of enzymatic activity withoutany chemical modification. Thus, ion-pairing is a useful method forincreasing the bioavailability of drugs and enhancing permeation ofcertain drugs. Dai et al., (Int. J. Pharm. 2007; 336: 58-66), Sun et al.(Int. J. Nanomed, 2011) prepared insulin hydrophobic ion paired complexusing different anionic surfactants like sodium lauryl sulphate,surfoplex, sodium deoxycholate and sodium oleate respectively in 1:6ratio.

In some embodiments, peptides disclosed herein can be modified byattaching one or more amphiphilic oligomers, for example, NobexTechnology. Creation of amphiphilic oligomers is accomplished by bindinga lipophilic alkyl unit to a hydrophilic polyethylene glycol (PEG) unit.The resulting oligomer is then covalently bound to a specific site orsites on the target molecule to facilitate the desired changes inproperties of the peptide. The two types of bonds are involved in thistechnology are hydrolyzable bond at the site of attachment, andnon-hydrolyzable bond to create a ‘micro-pegylated’ peptide. Methods forthis modification are described in, for example, Still J G. et al.Diabetes Metab. Res. Rev. 2002. In some embodiments, the peptidedisclosed herein can be modified by contacting with small hydrophobicorganic compounds non-covalently. In Emisphere'seligen™ technology. SeeSingh B et al. Oral Delivery of Therapeutic Macromolecules: APerspective Using the Eligen™ Technology; 2014.

Methods of Preparation of Pharmaceutical Compositions and Formulations

Disclosed herein are compositions comprising peptides or portion thereofcapable of biding CD40 and methods of their use. In some embodiments,the compositions disclosed herein are formulated for administration viaintracerebroventricular, intravenous, intradermal, intraperitoneal,oral, intramuscular, subcutaneous, intranasal, intracranial,intracelial, intracerebellar, intrathecal, transdermal, pulmonary, ortopical administration route.

The compositions provided herein can be prepared in a variety of waysdepending on the intended use of the compositions. For example, acomposition useful in practicing the technology herein may be a liquidcomprising a peptide disclosed herein in solution, in suspension, orboth (solution/suspension). The term “solution/suspension” refers to aliquid composition where a first portion of the active agent is presentin solution and a second portion of the active agent is present inparticulate form, in suspension in a liquid matrix. A liquid compositionalso includes a gel. The liquid composition may be aqueous or in theform of an ointment, salve, cream, or the like. An aqueous suspension orsolution/suspension useful for practicing the methods disclosed hereinmay contain one or more polymers as suspending agents. Useful polymersinclude water-soluble polymers such as cellulosic polymers andwater-insoluble polymers such as cross-linked carboxyl-containingpolymers. An aqueous suspension or solution/suspension of the presentdisclosure can be viscous or muco-adhesive, or both viscous andmuco-adhesive.

In some embodiments, the pharmaceutical compositions herein comprise apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans. The phrase “pharmaceutically acceptable carrier” as used hereinmeans a pharmaceutically acceptable material, composition or vehicle,such as a liquid or solid filler, diluent, excipient, solvent, media,encapsulating material, manufacturing aid (e.g., lubricant, talcmagnesium, calcium or zinc stearate, or steric acid), or solventencapsulating material, involved in maintaining the stability,solubility, or activity of, active agents in the compositions. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the patient.Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) excipients, such as cocoa butterand suppository waxes; (8) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (9)glycols, such as propylene glycol; (10) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol (PEG); (11) esters, such asethyl oleate and ethyl laurate; (12) agar; (13) buffering agents, suchas magnesium hydroxide and aluminum hydroxide; (14) alginic acid; (15)pyrogen-free water; (16) isotonic saline; (17) Ringer's solution; (19)pH buffered solutions; (20) polyesters, polycarbonates and/orpolyanhydrides; (21) bulking agents, such as polypeptides and aminoacids (22) serum components, such as serum albumin, HDL and LDL; (23)C2-C12 alcohols, such as ethanol; and (24) other non-toxic compatiblesubstances employed in pharmaceutical formulations. Release agents,coating agents, preservatives, and antioxidants can also be present inthe formulation. The terms such as “excipient”, “carrier”,“pharmaceutically acceptable carrier” or the like are usedinterchangeably herein. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin, andstill others are familiar to skilled artisans.

The compositions of the present disclosure can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like, including those adapted forthe following: (1) parenteral administration, for example, bysubcutaneous, intramuscular, intravenous or epidural injection as, forexample, a sterile solution or suspension, or sustained-releaseformulation; (2) topical application, for example, as a cream, ointment,or a controlled-release patch or spray applied to the skin; (3)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (4) ocularly; (5) transdermally; (6) transmucosally; or (7)nasally. The pharmaceutical compositions of the invention can beformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with free amino groups such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with free carboxyl groups such as those derived fromsodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The compositions disclosed herein can comprise a preservative. A“preservative” is a compound which can be added to the diluent toessentially reduce bacterial action in the reconstituted formulation,thus facilitating the production of a multi-use reconstitutedformulation, for example. Examples of potential preservatives includeoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkoniurn chloride (a mixture of alkylbenzyldimethylammoniumchlorides in which the alkyl groups are long-chain compounds), andbenzethonium chloride. Other types of preservatives include aromaticalcohols such as phenol, butyl and benzyl alcohol, alkyl parabens suchas methyl or propyl paraben, catechol, resorcinol, cyclohexanol,3-pentanol, and m-cresol. The most preferred preservative herein isbenzyl alcohol. One important purpose of the present formulation is tostabilize these peptide or analogue against spontaneous dimerization.This is preferably accomplished by formulating the peptides at low pHthat will prevent disulfide bond formation between two Cys residues.Such acid buffers are preferably biocompatible. Examples includecitrate, acetate, 2-(N-morpholino)ethanesulfonic acid (MES), or anysimilar buffer with a pK of about 5, wherein in the presence of thebuffer, the pH of the solution is <7.5 but preferably not below 3.0. Apreferred acidic formulation comprises citrate, preferably at about 25mM. The buffer is preferably supplemented with glycine (GIy) as anexcipient and bulking agent. A preferred concentration is about 50 mg/mlGIy. Another advantage of GIy is that it is an accepted excipient forintravenous infusion or injection in humans. Other amino acids orcompounds can be used in place of GIy. Examples of desirable acids orcombinations are citrate+acetate, acetate and Tris, and the like.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection.

If the compositions of the disclosure are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity preferablygreater than water are typically employed. Suitable formulationsinclude, without limitation, solutions, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like, which are, ifdesired, sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, preferably in combination with a solid or liquidinert carrier, is packaged in a mixture with a pressurized volatile(e.g., a gaseous propellant, such as freon) or in a squeeze bottle.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art.

If the compositions disclosed herein are to be formulated for intranasaladministration of a composition, the composition can be formulated in anaerosol form, spray, mist or in the form of drops. In particular,prophylactic or therapeutic agents for use according to the presentinvention can be conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant (e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas). In the case of a pressurized aerosol the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges (composed of, e.g., gelatin) for use in aninhaler or insufflator may be formulated containing a powder mix of thecompound and a suitable powder base such as lactose or starch.

If the compositions disclosed herein are to be formulated for oraladministration, compositions can be formulated orally in the form oftablets, capsules, cachets, gel caps, solutions, suspensions, and thelike. Tablets or capsules can be prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc,or silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well-known in the art. Liquidpreparations for oral administration may take the form of, but notlimited to, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring, and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated for slow release, controlled release, or sustained release ofa prophylactic or therapeutic agent(s).

The method of the disclosure may comprise pulmonary administration,e.g., by use of an inhaler or nebulizer, of a composition formulatedwith an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968,5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO97/44013, WO 98/31346, and WO 99/66903, each of which is incorporatedherein by reference their entireties. In a specific embodiment, anantibody of the invention, combination therapy, and/or composition ofthe invention is administered using Alkermes AIR pulmonary drug deliverytechnology (Alkermes, Inc., Cambridge, Mass.).

The method of the disclosure may comprise administration of acomposition formulated for parenteral administration by injection (e.g.,by bolus injection or continuous infusion). Formulations for injectionmay be presented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the disclosure may additionally comprise ofadministration of compositions formulated as depot preparations. Suchlong acting formulations may be administered by implantation (e.g.,subcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compositions may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the disclosure encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the disclosure also provides that one or more of thepeptides or pharmaceutical compositions of the disclosure is packaged ina hermetically sealed container such as an ampoule or sachetteindicating the quantity of the agent. In one embodiment, one or more ofthe peptides, or pharmaceutical compositions of the disclosure issupplied as a dry sterilized lyophilized powder or water freeconcentrate in a hermetically sealed container and can be reconstituted(e.g., with water or saline) to the appropriate concentration foradministration to a subject. Preferably, one or more of the peptides orpharmaceutical compositions of the disclosure is supplied as a drysterile lyophilized powder in a hermetically sealed container at a unitdosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg,at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least75 mg, or at least 100 mg. The lyophilized peptides or pharmaceuticalcompositions of the invention should be stored at between 2° C. and 8°C. in its original container and the peptides, or pharmaceuticalcompositions of the disclosure should be administered within 1 week,preferably within 5 days, within 72 hours, within 48 hours, within 24hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours,or within 1 hour after being reconstituted. In an alternativeembodiment, one or more of the peptides or pharmaceutical compositionsof the disclosure is supplied in liquid form in a hermetically sealedcontainer indicating the quantity and concentration of the agent.Preferably, the liquid form of the administered composition is suppliedin a hermetically sealed container at least 0.25 mg/ml, more preferablyat least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml.The liquid form should be stored at between 2° C. and 8° C. in itsoriginal container.

The pharmaceutical compositions can be administered in various ways,depending on the preference for local or systemic treatment, and on thearea to be treated. Administration may be done topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip orintraperitoneal, subcutaneous, subdural, intramuscular or intravenousinjection, or via an implantable delivery device. Formulations fortopical administration may include, but are not limited to, lotions,ointments, gels, creams, suppositories, drops, liquids, sprays andpowders Conventional pharmaceutical carriers, aqueous, powder or oilybases, thickeners and the like may be necessary or desirable.Compositions for oral administration include powders or granules,suspensions or solutions in water or nonaqueous media, sachets, capsulesor tablets. Thickeners, diluents, flavorings, dispersing aids,emulsifiers or binders may be desirable. Formulations for parenteraladministration may include, but are not limited to, sterile solutions,which may also contain buffers, diluents and other suitable additives.Formulations for implantable delivery devices may similarly include, butare not limited to, sterile solutions, which may also contain buffers,diluents and other suitable additives.

In some embodiments, a composition as described above is in apharmaceutically acceptable medium suitable for administration to arecipient subject. Pharmaceutically acceptable mediums suitable foradministration to a subject are known in the art. In some embodiments,compositions disclosed herein can be conveniently provided as sterileliquid preparations, e.g., isotonic aqueous solutions, suspensions,emulsions, dispersions, or viscous compositions, which may be bufferedto a selected pH. Liquid preparations are normally easier to preparethan gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection (e.g., intravenous injection).Viscous compositions, on the other hand, can be formulated within theappropriate viscosity range to provide longer contact periods withspecific tissues. Liquid or viscous compositions can comprise carriers,which can be a solvent or dispersing medium containing, for example,water, saline, phosphate buffered saline, polyol (for example, glycerol,propylene, glycol, liquid polyethylene glycol, and the like) andsuitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating thecompositions disclosed herein in the required amount of the appropriatesolvent with various amounts of the other ingredients, as desired. Suchcompositions may be in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose,dextrose, or the like. The compositions can also be lyophilized. Thecompositions can contain auxiliary substances such as wetting,dispersing, or emulsifying agents (e.g., methylcellulose), pH bufferingagents, gelling or viscosity enhancing additives, preservatives,flavoring agents, colors, and the like, depending upon the route ofadministration and the preparation desired. Standard texts, such as“Remington's Pharmaceutical Science”, 17th edition, 1985, incorporatedherein by reference, may be consulted to prepare suitable preparations,without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, may be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. The compositions can be isotonic, i.e., they can have the sameosmotic pressure as blood and lacrimal fluid. The desired isotonicity ofthe compositions herein may be accomplished using sodium chloride, orother pharmaceutically acceptable agents such as dextrose, boric acid,sodium tartrate, propylene glycol or other inorganic or organic solutes.Sodium chloride is preferred particularly for buffers containing sodiumions.

Parenteral dosage forms of the compositions can also be administered toa subject by various routes, including, but not limited to subcutaneous,intravenous (including bolus injection), intramuscular, andintraarterial. Since administration of parenteral dosage forms typicallybypasses the subject's natural defenses against contaminants, parenteraldosage forms are preferably sterile or capable of being sterilized priorto administration to a subject. Examples of parenteral dosage formsinclude, but are not limited to solutions ready for injection, dryproducts ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection,controlled-release parenteral dosage forms, and emulsions. Suitablevehicles that can be used to provide parenteral dosage forms of thedisclosure are well known to those skilled in the art. Examples include,without limitation: sterile water; water for injection USP; salinesolution; glucose solution; aqueous vehicles such as but not limited tosodium chloride injection, Ringer's injection, dextrose Injection,dextrose and sodium chloride injection, and lactated Ringer's injection;water-miscible vehicles such as, but not limited to ethyl alcohol,polyethylene glycol, and propylene glycol; and non-aqueous vehicles suchas, but not limited to corn oil, cottonseed oil, peanut oil, sesame oil,ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compositions provided herein can be packaged in a pressurized aerosolcontainer together with suitable propellants, for example, hydrocarbonpropellants like propane, butane, or isobutane with conventionaladjuvants. Compositions can also be administered in a non-pressurizedform such as in a nebulizer or atomizer. Compositions can also beadministered directly to the airways in the form of a dry powder, forexample, by use of an inhaler. Suitable powder compositions include, byway of illustration, powdered preparations of a composition comprising apeptide disclosed herein thoroughly intermixed with lactose, or otherinert powders acceptable for intrabronchial administration. The powdercompositions can be administered via an aerosol dispenser or encased ina breakable capsule which can be inserted by the subject into a devicethat punctures the capsule and blows the powder out in a steady streamsuitable for inhalation. The compositions can include propellants,surfactants, and co-solvents and can be filled into conventional aerosolcontainers that are closed by a suitable metering valve.

Aerosols for the delivery to the respiratory tract are known in the art.See for example, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569(1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115(1995); Gonda, I. “Aerosols for delivery of therapeutic and diagnosticagents to the respiratory tract,” in Critical Reviews in TherapeuticDrug Carrier Systems, 6:273-313 (1990); Anderson et al, Am. Rev. Respir.Dis., 140: 1317-1324 (1989)) and have potential for the systemicdelivery of peptides and proteins as well (Patton and Platz, AdvancedDrug Delivery Reviews, 8: 179-196 (1992)); Timsina et. al, Int. J.Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol. Market,4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., AerosolSci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10(1989)); Rudt, S, and R. H. Muller, J. Controlled Release, 22: 263-272(1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858(1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. andPlatz, R, Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug.Del. Rev., 5: 107-132 (1990); Patton, J. S., et al, Controlled Release,28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology(1996); Niven, R. W., et al., Pharm. Res., 12(9); 1343-1349 (1995); andKobayashi, S., et al., Pharm. Res., 13(1): 80-83 (1996), contents of allof which are herein incorporated by reference in their entirety.

The formulations of the compositions disclosed herein further encompassanhydrous pharmaceutical compositions and dosage forms comprising thedisclosed compounds as active ingredients, since water can facilitatethe degradation of some compounds. For example, the addition of water(e.g., 5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long-term storage in order to determine characteristics suchas shelf life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles & Practice, 379-80 (2nded., Marcel Dekker, NY, N.Y.: 1995). Anhydrous pharmaceuticalcompositions and dosage forms of the disclosure can be prepared usinganhydrous or low moisture containing ingredients and low moisture or lowhumidity conditions. Pharmaceutical compositions and dosage forms thatcomprise lactose and at least one active ingredient that comprises aprimary or secondary amine are preferably anhydrous if substantialcontact with moisture and/or humidity during manufacturing, packaging,and/or storage is expected. Anhydrous compositions are preferablypackaged using materials known to prevent exposure to water such thatthey can be included in suitable formulary kits. Examples of suitablepackaging include, but are not limited to hermetically sealed foils,plastics, unit dose containers (e.g., vials) with or without desiccants,blister packs, and strip packs.

In some embodiments of the aspects described herein, the compositionscan be administered to a subject by controlled- or delayed-releasemeans. Ideally, the use of an optimally designed controlled-releasepreparation in medical treatment is characterized by a minimum of drugsubstance being employed to cure or control the condition in a minimumamount of time. Advantages of controlled-release formulationsinclude: 1) extended activity of the active agent; 2) reduced dosagefrequency; 3) increased patient compliance; 4) usage of less total drug;5) reduction in local or systemic side effects; 6) minimization of drugaccumulation; 7) reduction in blood level fluctuations; 8) improvementin efficacy of treatment; 9) reduction of potentiation or loss of drugactivity; and 10) improvement in speed of control of diseases orconditions. (Kim, Chemg-ju, Controlled Release Dosage Form Design, 2(Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-releaseformulations can be used to control a compound of formula (I)'s onset ofaction, duration of action, plasma levels within the therapeutic window,and peak blood levels. In particular, controlled- or extended-releasedosage forms or formulations can be used to ensure that the maximumeffectiveness of a compound of formula (I) is achieved while minimizingpotential adverse effects and safety concerns, which can occur both fromunder-dosing a drug (i.e., going below the minimum therapeutic levels)as well as exceeding the toxicity level for the drug.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the compositionsdescribed herein. Examples include, but are not limited to thosedescribed in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598, 123;4,008,719; 5674,533; 5,059,595; 5,591,767; 5, 120,548; 5,073,543;5,639,476; 5,354,556; 5,733,566; and 6,365,185 B 1, each of which isincorporated herein by reference in their entireties. These dosage formscan be used to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS). (Alza Corporation, Mountain View, Calif. USA)), multilayercoatings, microparticles, liposomes, or microspheres or a combinationthereof to provide the desired release profile in varying proportions.Additionally, ion exchange materials can be used to prepare immobilized,adsorbed salt forms of the disclosed compounds and thus effectcontrolled delivery of the drug. Examples of specific anion exchangersinclude, but are not limited to Duolite. A568 and Duolite. AP143(Rohm&Haas, Spring House, Pa. USA). In some embodiments, compositionsdescribed herein can be administered to a subject by sustained releaseor in pulses. Pulse therapy is not a form of discontinuousadministration of the same amount of a composition over time, butcomprises administration of the same dose of the composition at areduced frequency or administration of reduced doses. Sustained releaseor pulse administrations are particularly preferred when the disorderoccurs continuously in the subject, for example where the subject hascontinuous or chronic symptoms of an inflammatory disease. Each pulsedose can be reduced and the total amount of a peptide disclosed hereincan be administered over the course of treatment to the subject isminimized.

The interval between pulses, when necessary, can be determined by one ofordinary skill in the art. Often, the interval between pulses can becalculated by administering another dose of the composition when thecomposition or the active component of the composition is no longerdetectable in the subject prior to delivery of the next pulse. Intervalscan also be calculated from the in vivo half-life of the composition.Intervals can be calculated as greater than the in vivo half-life, or 2,3, 4, 5 and even 10 times greater the composition half-life. Variousmethods and apparatus for pulsing compositions by infusion or otherforms of delivery to the patient are disclosed in U.S. Pat. Nos.4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, and carriersinclude aqueous isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, preservatives,liposomes, microspheres and emulsions. The compositions described hereininclude, but are not limited to therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions contain a physiologically tolerable carrier together withan active agent as described herein, dissolved or dispersed therein asan active ingredient. In one embodiment, the therapeutic composition isnot immunogenic (e.g., allergenic) when administered to a mammal orhuman patient for therapeutic purposes. As used herein, the terms“pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a mammal without theproduction of undesirable physiological effects such as nausea,dizziness, gastric upset and the like. A pharmaceutically acceptablecarrier will not promote the raising of an immune response to an agentwith which it is admixed, unless so desired. The preparation of apharmacological composition that contains active ingredients dissolvedor dispersed therein is well understood in the art and need not belimited based on formulation. The active ingredient can be mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient and in amounts suitable for use in the therapeuticmethods described herein. Suitable excipients include, for example,water, saline, dextrose, glycerol, ethanol or the like and combinationsthereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents and the like which enhance the effectiveness of theactive ingredient. The compositions described herein can includepharmaceutically acceptable salts of the components therein.

Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active agent used in the methodsdescribed herein that will be effective in the treatment of a particulardisorder or condition will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques.

While any suitable carrier known to those of ordinary skill in the artcan be employed in the pharmaceutical compositions provided herein, thetype of carrier will vary depending on the mode of administration.Compositions can be formulated for any appropriate manner ofadministration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) can also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109. Such compositions can also comprise buffers(e.g., neutral buffered saline or phosphate buffered saline),carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol,proteins, polypeptides or amino acids such as glycine, antioxidants,chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminumhydroxide) and/or preservatives. Alternatively, compositions asdescribed herein can be formulated as a lyophilizate. Compounds can alsobe encapsulated within liposomes using well known technology. Thecompositions described herein can be administered as part of a sustainedrelease formulation (i.e., a formulation such as a capsule or spongethat effects a slow release of compound following administration). Suchformulations can generally be prepared using well known technology andadministered by, for example, oral, rectal or subcutaneous implantation,or by implantation at the desired target site. Sustained-releaseformulations can contain a polypeptide, polynucleotide dispersed in acarrier matrix and/or contained within a reservoir surrounded by a ratecontrolling membrane. Carriers for use within such formulations arebiocompatible, and can also be biodegradable; preferably the formulationprovides a relatively constant level of active component release. Theamount of active compound contained within a sustained releaseformulation depends upon the site of implantation, the rate and expectedduration of release and the nature of the condition to be treated orprevented.

In some embodiments, the pharmaceutical composition is contained in animplantable pump. The pump can be designed to deliver to the intendedsite of action, at the required rate of administration, and in theproper therapeutic dose. One example of such pump includes but is notlimited to the commercially available, the Alzet osmotic mini pump todelivering drugs at a controlled rate and dose over extended periods.

In some embodiments, the peptides of the compositions disclosed hereincan be contained in a continuous flow pump. The delivery mechanism ofone such pump is based on the expansion of Freon gas at 37° C. thatpushes a diaphragm “plunger/pusher” plate. Usually, the pump reservoiris implanted subcutaneously and is connected to a catheter to deliverthe therapeutic molecules. In some embodiments, the peptides can becontained in a programmable pump. Programmable pumps includeelectromechanical pumps of the peristaltic type, powered by batteries.Their built-in electronics can be remotely controlled from an externalprogramming unit. An example is the SynchroMed system (Medtronic Inc.).The infusion can be programmed in various modes: continuous hourlyinfusions, repeated bolus infusions with a specified delay, multipledoses over a programmed interval, or a single bolus infusion. In someembodiments, the pharmaceutical composition comprising peptidesdisclosed herein is contained in a syringe, including a blunt tipsyringe for injection to the target site. Microspheres can be implantedstereotactically in the target site.

In certain embodiments, the compositions disclosed herein can beformulated in liposomes, for example to improve stability and promotedelivery. As used herein, the term “liposome” refers to a vesicularstructure having lipid-containing membranes enclosing an aqueousinterior. In cell biology, a vesicular structure is a hollow, lamellar,spherical structure, and provides a small and enclosed compartment,separated from the cytosol by at least one lipid bilayer. Liposomes canhave one or more lipid membranes. Oligolamellar large vesicles andmultilamellar vesicles have multiple, usually concentric, membranelayers and are typically larger than 100 nm. Liposomes with severalnonconcentric membranes, i.e., several smaller vesicles contained withina larger vesicle, are termed multivesicular vesicles. Liposomes canfurther comprise one or more additional lipids and/or other componentssuch as sterols, e.g., cholesterol. Additional lipids can be included inthe liposome compositions for a variety of purposes, such as to preventlipid oxidation, to stabilize the bilayer, to reduce aggregation duringformation or to attach ligands onto the liposome surface. Any of anumber of additional lipids and/or other components can be present,including amphipathic, neutral, cationic, anionic lipids, andprogrammable fusion lipids. Such lipids and/or components can be usedalone or in combination. One or more components of the liposome cancomprise a ligand, e.g., a targeting ligand.

Liposome compositions can be prepared by a variety of methods that areknown in the art. See e.g., U.S. Pat. No. 6,339,069B1. Niosomes arenon-phospholipid based synthetic vesicles that have properties andfunction like liposomes. Liposomes are vesicular structures with anaqueous core surrounded by a hydrophobic lipid membrane created byextrusion of phospholipids and known in the art to be used for drugdelivery purposes. In some embodiments of the compositions and methodsdescribed herein, the peptide is encapsulated in a liposome. Liposomescan vary in size from 15 nm to 100μπι and are contemplated to haveeither a single layer (uni-lamellar), or multiple phospholipid bilayermembranes (mutilamellar structure). In one aspect, the peptide can beencapsulated in a niosome, a non-phospholipid-based synthetic vesicle

In some embodiments, the compositions disclosed herein are formulatedusing micelles formed from lipid-associated peptides disclosed herein,e.g., peptides of the present disclosure conjugated to at least oneamphiphilic carrier, in which the micelles have an average diameter ofless than about 100 nm, preferably. More preferred embodiments providemicelles having an average diameter less than about 50 nm, and even morepreferred embodiments provide micelles having an average diameter lessthan about 100 nm, or even less than about 20 nm. As used herein,“micelles” are a particular type of molecular assembly in whichamphipathic molecules are arranged in a spherical structure such thatall hydrophobic portions on the molecules are directed inward, leavingthe hydrophilic portions in contact with the surrounding aqueous phase.The converse arrangement exists if the environment is hydrophobic.

In one aspect, the peptides of the present disclosure are encapsulatedin a micelle. Micelles are spherical aggregates of amphiphilic moleculesdispersing in water with their hydrophilic head groups on the surface ofthe sphere, and their hydrophobic tails collected inside. An importantproperty of micelles is their ability to increase the solubility andbioavailability of poorly soluble pharmaceuticals. The amphiphilicmolecules in micelles are in constant exchange with those in the bulksolution. On the other hand, polymeric micelles, also known aspolymersomes, are self-assembled polymer shells composed of blockcopolymer amphiphiles such as polyethylene glycol-polylactic acid(PEG-PLA) and PEG-polycaprolactone (PEG-PCL). Polymeric micelles differfrom nanoparticles that are either more solid or monolithic(nanospheres) or contain an oily or aqueous core and are surrounded by apolymer shell (nanocapsules). However, in practice, polymeric micellesalso be referred to as nanoparticle or nanocarriers because of theirparticle size.

Accordingly, in some embodiments the peptides of the compositionsdisclosed herein is encapsulated in a nanoparticle. In some embodiment,the peptide disclosed herein can be encapsulated in a microcapsule or amicrosphere, which are free flowing powders consisting of sphericalparticles of 2 millimeters or less in diameter, usually 500 microns orless in diameter. As used herein, the term “nanoparticle” refers to aparticle having a size between 1 and 1000 nm which can be manufacturedfrom artificial or natural macromolecular substances. To suchnanoparticles can be bound drugs or other biologically active materialsby covalent, ionic or adsorptive linkage, or the latter can beincorporated into the material of the nanoparticles. Nanoparticles mayor may not exhibit size-related properties that differ significantlyfrom those observed in fine particles or bulk materials. Nanoparticlesprovide improved bioavailability by enhancing aqueous solubility,increasing resistance time in the body (increasing half-life forclearance/increasing specificity for its cognate receptors and targetingdrug to specific location in the body (its site of action). This resultsin concomitant reduction in quantity of the drug required and dosagetoxicity, enabling the safe delivery of toxic therapeutic drugs andprotection of non-target tissues and cells from severe side effects.Non-limiting examples of nanoparticles include solid lipid nanoparticles(comprise lipids that are in solid phase at room temperature andsurfactants for emulsification, the mean diameters of which range from50 nm to 1000 nm for colloid drug delivery applications), liposomes,nanoemulsions (oil-in-water emulsions done on a nano-scale), albuminnanoparticles, and polymeric nanoparticles. Nanoparticles can be surfacecoated to modulate their stability, solubility, and targeting. A coatingthat is multivalent or polymeric confers high stability. A non-limitingexample includes coating with hydrophilic polymer such as polyethyleneglycol or ploysorbate-80.

Methods of making nanoparticles encompassing therapeutic molecules areknown in the art. See for example, U.S. Pat. No. 6,207,195B1,US20080311214A1, US20080311214A1, Tan et al, 2010. Nanoparticles aresolid matrix colloidal particles with diameters ranging from 1-100 nmformed using various polymers like degradable starch, dextran, chitosan,microcrystalline cellulose (MCC), hydroxypropyl cellulose (HPC),hydroxypropyl ethylcellulose (HPMC), carbomer, and wax-like starch,gelatin polymers. In these carrier systems, the drug can be loaded viaeither incorporation with the system or its adsorption on theparticulate system. The encapsulating nanoparticle can be, for example,solid-lipid nanoparticles (SLNs), polymeric nanoparticles, oroil-in-water nanoemulsions. Solid-lipid nanoparticles aresurfactant-stabilized aqueous colloidal dispersions of lipidnanoparticles that solidify upon cooling. They contain a lipid phasedispersed in an aqueous environment. Polymeric nanoparticles are solidcolloidal particles created from polymeric systems. These nanoparticlesare made from biocompatible polymers that encapsulate or adsorb drugsfor prolonged release. Nanoemulsions are oil-in-water (OAV) orwater-in-oil (W/O) formulations made with edible or otherwisepharmaceutically acceptable oils, surface-active agents (surfactants),and water, where the diameter of the inner phase is reduced to nanometerlength scale. The versatility of nanoemulsions is based on the differenttypes of oils and surface modifiers that can be used. For instance, oilsthat are rich in omega-3 polyunsaturated fatty acids (PUFA) can play avery important role in overcoming biological barriers, including theblood brain barrier.

For effective targeting, the liposomes and nanoparticles encapsulatingthe peptides disclosed herein can be further linked with and/or coatedwith other agents. One example of such an agent can be an antibodybinding fragment such as Fab, F(ab′)2, Fab′ or a single antibody chainpolypeptide which binds to a receptor molecule present on a target cellof interest, (e.g., immune cell expressing CD40 or a neoplastic cellexpressing CD40 on its surface). In some embodiments, the nanoparticlesencapsulating the peptides of the compositions and methods disclosedherein can be coated with poly(ethylene glycol) or polysorbate 80 oralbumin or its functional groups. PEG-containing surfactants,poly(oxy-ethylene)-poly(oxy-propylene) can also be used for coatingnanoparticles. Polysorbate 80-coated poly(n-butylcyanoacrilate)nanoparticles have been formulated by emulsion polymerization method totarget selectively rivastigmine or tacrine. Nanoparticles and liposomescan also be linked to carrier peptides for examples TAT, to improvetheir lipophilicity.

In some embodiments, the peptides of the compositions and methodsdisclosed herein can be fused to a carrier peptide, for example fortransdermal administration. See for example, US20080305989A1,EP2857033A2

In some embodiments, the compositions of the present disclosure areformulated for intranasal administration. Therefore, in order to enhancethe absorption of a therapeutic drug or agent into the olfactoryneurons, the drug or agent should be capable of at least partiallydissolving in the fluids that are secreted by the mucous membrane thatsurround the cilia of the olfactory receptor cells of the olfactoryepithelium. Therefore the therapeutic peptide can be linked to a carrierthat increases its dissolution within nasal secretions. Non-limitingexamples of such carriers include GM-1 ganglioside, phosphotidylserine(PS), and emulsifiers such as polysorbate 80. Linkage with lipophiliccarriers such as gangliosides or phosphotidylserine can improve theadsorption of the therapeutic drug into the olfactory neurons andthrough the olfactory epithelium. In some embodiments, the compositionsof the present disclosure comprises a ganglioside or aphosphatidylserine (e.g., composition comprising a peptide disclosedherein formulated for intranasal administration).

In some embodiments, the peptides of the present disclosure can beencapsulated in nanoparticles, liposomes, micelles, microspheres,niosomes, cyclodextrin-inclusion complexes, or nanoemulsions. Thenanoparticles can be coated with polymers such as polyethyleneglycol-polylactic acid (PEG-PLA) or Chitosan (CS). Methods forpreparation of (PEG-PLA) nanoparticles are well known in the art. Thechitosan nanoparticles can be complexed with cyclodextrins. In someembodiments, the compositions of the present disclosure further comprisecyclodextrin. Use of cyclodextrin for delivery of peptide compositionsis known in the art (US20120302505A1) The term “cyclodextrins” refers tocyclic oligosaccharides, like α-, β- and γ-cyclodextrin and theirderivatives, preferably β-cyclodextrin and its derivatives, preferablymethylated β-cyclodextrin, with a degree of CH3-substitution between 0.5and 3.0, more preferably between 1.7 and 2.1. The term “saccharides”refers to disaccharides, like lactose, maltose, saccharose and alsorefers to polysaccharides, like dextrans, with an average molecularweight between 10,000 and 100,000, preferably 40,000 and 70,000. Theterm “sugar alcohols” refers to mannitol and sorbitol. In someembodiments, the compositions of the present disclosure furthercomprises saccharides selected from the group consisting ofcyclodextrins, disaccharides, polysaccharides and combinations thereof.Peptides can be encapsulated in carriers, like cyclodextrins inclusioncomplexes containing a hydrophobic core and a hydrophilic shell whichcan help improve upon the solubility problems.

The compositions can be dispensed intranasally as a powdered or liquidnasal spray, nose drops, a gel or ointment, injection or infusioncontained in a tube or catheter, by syringe, by pledge, or by submucosalinfusion. Also the composition can made viscous using vehicles such asnatural gums, methylcellulose and derivatives, acrylic polymers(carbopol) and vinyl polymers (polyvinylpyrrolidone). Many otherexcipients, known in the pharmaceutical literature, can be added, suchas preservatives, surfactants, co-solvents, adhesives, antioxidants,buffers, viscosity enhancing agents, and agents to adjust the pH or theosmolarity.

Nasal powder compositions can be made by mixing the active agent and theexcipient, both possessing the desired particle size. Other methods tomake a suitable powder formulation can be selected. Firstly, a solutionof the active agent and the cyclodextrin and/or the other saccharideand/or sugar alcohol is made, followed by precipitation, filtration andpulverization. It is also possible to remove the solvent by freezedrying, followed by pulverization of the powder in the desired particlesize by using conventional techniques, known from the pharmaceuticalliterature. The final step is size classification for instance bysieving, to get particles that are less than 100 microns in diameter,preferably between 50 and 100 microns in diameter. Powders can beadministered using a nasal insufflator. Powders may also be administeredin such a manner that they are placed in a capsule. The capsule is setin an inhalation or insufflation device. A needle is penetrated throughthe capsule to make pores at the top and the bottom of the capsule andair is sent to blow out the powder particles. Powder formulation canalso be administered in a jet-spray of an inert gas or suspended inliquid organic fluids. In some embodiments, the composition forintranasal administration can be adapted for aerosolization andinhalation. The composition can be administered nasally via pressurizedaerosol, aqueous pump spray or other standard methods known to thoseskilled in the art.

The compositions of present disclosure can be administered in the formof spray in a non-pressurized aerosol device, for example a Pfeifferpump. In some embodiments, the compositions can be contained in asyringe, catheter, an inhaler, a nebulizer, a nasal spray pump, a nasalirrigation pump, or a nasal lavage pump. Non-limiting example forintranasal administration of liquid formulation can include (a)delivering drops with a drop pipette, (b) rhinyle catheter and squirttube which involves inserting the tip of a fine catheter or micropipetteto the desired area under visual control and squirt the liquid into thedesired location, (c) squeeze bottles which involve squeezing a partlyair-filled plastic bottle, to deliver the atomized drug from a jetoutlet, (d) metered-dose spray pumps which deliver a unit dose of drugper use, (e) single- and duo-dose spray devices which is used for asingle administration of a unit dose of drug, (f) nasal pressurizedmetered-dose inhalers delivers a nasal aerosol preparation, and (g)powered nebulizers and atomizers to administer drug in the form of amist. Non-limiting examples for intranasal administration of liquidformulations can include nasal powder inhalers (e.g., RhinocortTurbuhaler®; BiDose™/Prohaler™) from Pfeiffer/Aptar), nasal powdersprayers (e.g., Fit-lizer™ device, Unidose-DP™), and nasal powderinsufflators (e.g., Bi-Directional™ nasal delivery, Optinose).

Lyophilized Composition:

The compositions of the present disclosure can be provided in alyophilized form. The composition in a lyophilized form can furthercomprise a lyoprotectant. A “lyoprotectant” is a molecule which, whencombined with a protein or peptide of interest, significantly preventsor reduces chemical and/or physical instability of the protein orpeptide upon lyophilization and subsequent storage. Exemplarylyoprotectants include sugars such as sucrose or trehalose; an aminoacid such as monosodium glutamate or histidine; a methylamine such asbetaine; a lyotropic salt such as magnesium sulfate; a polyol such astrihydric or higher sugar alcohols, e.g., glycerin, erythritol,glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol;polyethylene glycol; pluronics; and combinations thereof. The preferredlyoprotectant is a non-reducing sugar, such as trehalose or sucrose. Thelyoprotectant is added to the pre-lyophilized formulation in a“lyoprotecting amount” which means that, following lyophilization of thepeptide in the presence of the lyoprotecting amount of thelyoprotectant, the peptide essentially retains its physical and chemicalstability and integrity upon lyophilization and storage. The compositionin a lyophilized form can comprise a bulking agent. A “bulking agent” isa compound which adds mass to the lyophilized mixture and contributes tothe physical structure of the lyophilized cake (e.g., facilitates theproduction of an essentially uniform lyophilized cake which maintains anopen pore structure). Exemplary bulking agents include mannitol,glycine, polyethylene glycol and xorbitol.

Reconstitution of the Lyophilized Formulation

At the desired stage, typically when it is time to administer thepeptide to a subject, the lyophilized formulation may be reconstitutedwith a diluent such that the peptide concentration in the reconstitutedformulation is at least 10 mg/mL, for example from about 10 mg/mL toabout 1000 mg/mL, more preferably from about 50 mg/mL to about 500mg/mL, and most preferably from about 100 mg/mL to about 500 mg/mL. Suchhigh peptide concentrations in the reconstituted formulation areconsidered to be particularly useful where subcutaneous delivery of thereconstituted formulation is intended. However, for other routes ofadministration, such as intravenous (i.v.) administration, lowerconcentrations of the peptide in the reconstituted formulation may bedesired (for example from about 1-100 mg/mL, or from about 5-50 mg/mLpeptide in the reconstituted formulation). In certain embodiments, thepeptide concentration in the reconstituted formulation is significantlyhigher than that in the pre-lyophilized formulation. For example, thepeptide concentration in the reconstituted formulation may be about 2-40times, preferably 3-10 times and most preferably 3-6 times (e.g., atleast three fold or at least four fold) that of the pre-lyophilizedformulation.

Reconstitution generally takes place at a temperature of about 25° C. toensure complete hydration, although other temperatures may be employedas desired. The time required for reconstitution will depend, forexample, on the type of diluent, amount of excipient(s) and peptide. The“diluent” of interest is one which is pharmaceutically acceptable (safeand non-toxic for administration to a human) and is useful for dilutinga peptide in solution or the preparation of a reconstituted formulation.Exemplary diluents include sterile water, bacteriostatic water forinjection (BWFI), a pH buffered solution (e.g. phosphate-bufferedsaline, PBS), sterile saline solution, Ringer's solution or dextrosesolution. The diluent optionally contains a preservative. Exemplarypreservatives have been described above, with aromatic alcohols such asbenzyl or phenol alcohol being the preferred preservatives. The amountof preservative employed is determined by assessing differentpreservative concentrations for compatibility with the peptide andpreservative efficacy testing. For example, if the preservative is anaromatic alcohol (such as benzyl alcohol), it can be present in anamount from about 0.1-2.0% and preferably from about 0.5-1.5%, but mostpreferably about 1.0-1.2%. Preferably, the reconstituted formulation hasless than 6000 particles per vial which are >10 μm in size.

In general, the composition, comprising the peptides, may be packaged insterile form in a sterile container such as a vial, syringe (forinjection) etc. In some embodiments, the composition has a shelf life inpackaged form of at least 2, 3 or 4 days, up to 1, 2 or 3 weeks or onemonth, or in some embodiments a shelf life in packaged form of at leastone or two months up to 6 months or one to two years, or more, at atemperature of 37° C., or at room temperature. “Shelf life” has itsconventional meaning in the art and refers to the time period duringwhich the active agent retains sufficient activity for accurate andeffective administration thereof, and the formulation retains sufficientstability (e.g., a single phase solution remains as a single phasesolution) for accurate and effective administration thereof at therecited temperature conditions.

Methods of Use Assays

The peptides of the present disclosure selectively bind CD40 protein. Insome embodiments, the CD40 protein is expressed on a surface of a cell(e.g., an immune cell, a non-immune cell, a cancer cell). It will bewell appreciated by those skilled in the art that the peptides orportion thereof disclosed herein can bind CD40 protein in vitro, (forexample when used in a cell culture system), ex vivo, and in vivo. Insome embodiments, peptides of the present disclosure can be used todetect CD40 expressing cells. In some embodiments, the peptide isconjugated to a detectable agent, for example, fluorescent agent,chemiluminescent agent, radioistope, or enzyme-substrate agent such asalkaline phosphatase. An assay for detecting a cell expressing CD40 in asample comprises, for example, a) contacting the sample with a peptideof the present disclosure under condition suitable to allow binding ofthe peptide with CD40 on the cell surface; b) washing the unboundpeptide; and c) detecting the peptide bound to cells. The peptide can bedetected, for example, by screening for the detectable agent conjugatedto the peptide. The presence of detectable agent indicates presence ofCD40 expressing cell. In some embodiments, a secondary detection agent(e.g., an antibody binding the peptide disclosed herein) can be used todetect the peptide bound to CD40 expressing cell. In some embodiments,the peptide useful for such assays can be immobilized onto a solidsurface. The solid surface can be in the form of tubes, beads, discs,silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane,nitrocellulose membrane, nylon membrane, other porous membrane,non-porous membrane, e.g., plastic, polymer, perspex, silicon, amongstothers, a plurality of polymeric pins, or a plurality of microtitrewells, or any other surface suitable for immobilizing polypeptide orpolynucleotides.

Activated immune cells expressing CD40 are associated with risk ofdeveloping inflammatory diseases, autoimmune disease or cancer.Accordingly, in some embodiments, the peptides disclosed herein can beused to identify patients at risk of developing inflammatory diseases,autoimmune disease or a cancer. A sample obtained from candidatepatients can be assayed for presence of CD40 expressing cells using, forexample, an assay described above. In some embodiments, the sample canbe screened for a specific type of cell expressing CD40 alone or incombination with other surface markers specific for that cell type. Forexample, a sample can be assayed for a population of activated T-cell byusing the peptides disclosed herein to detect expression of CD40 aloneor using the peptides disclosed herein in combination with moleculesdetecting activated T-cell specific surface proteins such as CD25, CD38,CD69 and the like. In another embodiment, a sample can be assayed toidentify a CD40 expressing cancer cell using the peptides disclosedherein alone or by using the peptides disclosed herein to detect theCD40 in combination with molecules detecting cancer markers on cellsurface. An increase in number of a specific type of cell (e.g.,activated T-cell) relative to the total cell population in a sample canbe indicative of a disease condition (e.g. inflammatory diseases,autoimmune disease or a cancer).

In some embodiments, the peptides disclosed herein can be used to screenfor molecules (e.g., small molecule, proteins, peptides) that bind CD40and modulate CD40 activity. The assay can be designed, for example, as acompetitive binding assay. The assay can be designed, to test theability of a test molecule to compete with the peptides disclosed hereinfor binding to a CD40 protein. The peptides disclosed herein useful forsuch an assay can be one that is fused to a detectable agent. In oneembodiment, the method comprises: a) contacting a cell or biologicalsample with a peptide disclosed herein in the presence or absence of atest agent under conditions which allow for their binding to CD40; b)washing the sample to remove any unbound peptides; and c) screening thesample for presence of detectable agent. In one embodiment, absence of adetectable agent, indicates the test molecule is able to compete withthe peptide for binding to a CD40 protein. The assay can be used, forexample, to screen for molecules that partially inhibit binding ofpeptides to the CD40 protein. Methods of competitive binding assay arewell known in the art. The test agents can be obtained using any of thenumerous approaches in combinatorial-library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam et al., 1997, Anticancer DrugDes.12:45). Examples of methods for the synthesis of molecular librariescan be found in the art, for example, in: DeWitt et al, 1993, Proc.Natl. Acad. USA 90:6909; Erb et al, 1994, Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al, 1994, J. Med. Chem. 37:2678; Cho et al, 1993,Science 261: 1303; Carrell et al, 1994, Angew. Chem. Int. Ed. Engl.33:2059; Carell et al, 1994, Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al, 1994, J. Med. Chem. 37: 1233. Libraries of compounds maybe presented in solution (e.g., Houghten, 1992, Biotechniques13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor,1993, Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409),spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al, 1992, Proc.Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990,Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al,1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991, J. Mol.Biol. 222:301-310; and Ladner supra).

In situations where “high-throughput” modalities are preferred, it istypical that new chemical entities with useful properties are generatedby identifying a chemical compound (called a “lead compound”) with somedesirable property or activity, creating variants of the lead compound,and evaluating the property and activity of those variant compounds. Thecurrent trend is to shorten the time scale for all aspects of drugdiscovery. In one embodiment, high throughput screening methods involveproviding a library containing a large number of compounds (candidatecompounds) potentially having the desired activity. Such “combinatorialchemical libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics. Assaysdisclosed herein are meant as examples of useful assays and it isunderstood that other assays or modifications of assays described aboveusing the peptides disclosed herein can be employed. Suitable assaytechniques are known to those skilled in the art and are disclosed forexample in Molecular Cloning: A Laboratory Manual, Sambrook, J.,Fritsch, E. F., and Maniatis, T, Cold Spring Harbor Laboratory Press;2nd Edition (December 1989). All referenced cited herein areincorporated herein in their entirety.

Methods of Immunosuppression and Treatment

The term “immunosuppression” as used herein relates to an inhibition inthe activation and/or maintenance of an immune response (for example,immune response directed towards a transplant). As used herein, the term“immune response” refers to the alteration in the reactivity of anorganism's immune system upon exposure to an antigen. The term “immuneresponse” encompasses but is not limited to one or both of the followingresponses: antibody production (e.g., humoral immunity), and inductionof cell-mediated immunity (e.g., cellular immunity including helper Tcell and/or cytotoxic T cell responses). “Immune response” generallyrefers to innate and acquired immune responses including, but notlimited to, both humoral immune responses (mediated by B lymphocytes)and cellular immune responses (mediated by T lymphocytes). An immuneresponse may be beneficial and lead to immunity against infectiouspathogens, or an immune response may be pathogenic and lead toautoimmune or hypersensitivity disease. Immune responses against foreignviruses, bacteria, fungi, parasites typically represent beneficialadaptive immune responses. Immune responses against self tissues,innocuous foreign objects (e.g., dust mite or pollen allergens, etc.),or tissue transplants represent examples of adverse maladaptive immuneresponses. The term “modulate,” “modulates” or “modulation” refers toenhancement (e.g., an increase) or inhibition (e.g., a reduction) in thespecified activity or suppression thereof. As it relates to the presentdisclosure, immunosuppression also refers to inhibition of an immuneresponse mediated by CD40 activity or one that is mediated byinteraction of CD40 with CD154.

CD40 is well known to play an important role in activation of immuneresponse. B cells are known to constitutively express the CD40 moleculeon the cells membrane (van Kooten C. et al, J. Leukoc. Biol. 2000).Stimulation of CD40 by CD154-expressing activated T cells, results in Bcells activation, proliferation and isotype switching. Recent studieshave indicated that CD40-CD154 interaction can upregulate costimulatorymolecules, activate APCs, and influence T-cell priming andT-cell-mediated effector CD40 activities include modulating cellsurvival (e.g., functions as cell survival signal), antibody production,antibody isotype switching, production of cytokines (e.g., IL-2, IL-6,IL-8, IL-12, TNF-α, IL-4, IL-5 and IL-10), metalloproteases (e.g.,MMP-I/collagenase and MMP-9/gelatinase B) and establishment of immunememory. CD40 activities further include modulating production ofproteins involved in cell-cell contact or adhesion (e.g., E-selectin,VCAM-1 and ICAM-1). This interaction can activate macrophages, naturalkiller (NK) cells, and endothelial cells. CD40 plays a significant rolein immune cell function and signaling, including B-cell and T-cellactivation by antigen presenting cells, such as macrophages anddendritic cells. CD40 activity stimulates antibody production, isotypeswitching, and establishment of memory. CD40 activity stimulatesproduction of cytokines, such as IL-2, IL-6, IL-8, IL-12, TNF-α, IL-4,IL-S and IL-10; and metalloproteases, such as MMP-I/collagenase andMMP-9/gelatinase B. CD40 activity stimulates production of proteinsinvolved in cell-cell contact or adhesion, such as E-selectin, VCAM-1and ICAM-1. CD40 recognition on target cells provides an activationpathway for NK cell cytotoxic activity. Thus, increasing or decreasingcell survival, antibody production, isotype switching, establishment ofmemory, production of cytokines, metalloproteases or proteins involvedin cell-cell contact or adhesion, NK cell cytotoxic activity, B-cellactivation, B-cell proliferation, T-cell activation, T-cellproliferation, macrophage activation, migration of immune cell can allbe effected or inhibited by contacting an appropriate cell expressingCD40 with the peptide of the present disclosure or a pharmaceuticalcomposition disclosed herein

Thus, the disclosure further provides methods of modulating cellsurvival, antibody production, isotype switching, establishment ofmemory, production of cytokines, metalloproteases or proteins involvedin cell-cell contact or adhesion, NK cell cytotoxic activity, B-cellactivation, B-cell proliferation, T-cell activation, T-cellproliferation, macrophage activation, or migration of an immune cell,comprising contacting a cell expressing CD40 with an effective amount ofa peptide disclosed herein. By “a cell expressing CD40” herein isintended any normal or malignant cells that express detectable levels ofthe CD40 antigen. The cell can be an immune cell, a nonimmune cell, or acancer cell, for example, lymphoma cell. Methods for detecting CD40expression in cells are well known in the art and include, but are notlimited to, PCR techniques, immunohistochemistry, flow cytometry,Western blot, ELISA, and the like. These methods allow for the detectionof CD40 mRNA, CD40 antigen and cell-surface CD40 antigen. Preferably,the CD40-expressing cells are cells that express detectable levels ofcell-surface CD40 antigen. The cell to be contacted can be informed bythe immune response to be modulated. For example, in embodiments relatedto B-cell mediated immune response, such as B-cell proliferation, B-cellactivation, the cell contacted with the peptides or compositionsdisclosed herein can be a B-cell. For example, in embodiments related toinhibiting activation of macrophage activation or production ofcytokines, the cell contacted with the peptides or compositionsdisclosed herein can be a macrophage. The cell to be contacted can beeasily determined by one of skill in the art.

Assays for monitoring immune responses are well known in the art and aredescribed above. Methods of immunosuppression can be practiced on asubject in order to achieve the effect in the subject. Accordingly, thedisclosure provides for a method for inducing immunosuppression in asubject in need thereof, the method comprising administering to thesubject an effective amount of a peptide of the present disclosure or apharmaceutical composition disclosed herein.

Compositions, methods, kits, and systems provided herein can be utilizedto prevent and/or treat an autoimmune disorder. The term “autoimmunedisorder”, “autoimmune disease”, “autoimmune condition”, and theirgrammatical equivalents as used herein can be used interchangeably. Insome cases, the tolerance vaccine provided herein can be crosslinked toautoantigenic peptides, autoantigens, or other cellular carriers andused as a tolerance therapy for an autoimmune disorder. In some cases,the cellular carrier is an apoptotic cellular carrier. In some cases,the cellular carrier is a syngeneic apoptotic cellular carrier.

The tolerizing vaccine as described herein can be used with a cellularcarrier (e.g., autoantigens, autoantigenic peptides, apoptotic cellularcarriers) to induce antigen-specific T cell tolerance for treatment ofan autoimmune condition. Without being bound by theory, the tolerizingvaccine with or without the carrier can be taken up, processed, andpresented in a tolerogenic manner by host splenic antigen presentingcells, thereby inducing regulatory T cells, and the secretion of immunesuppressive cytokines (e.g., IL-4, IL-10, IL-13, TGF-β).

Methods and composition of the present disclosure can be employed totreat a CD40 associated disorder. The disclosure therefore also providesmethods of treating a CD40 associated disorder. As used herein, the term“CD40 associated disorder” means any undesirable physiological conditionor pathological disorder in which modulating a CD40 activity (e.g.,binding CD154 or activation of immune response mediated by CD40 mayimprove or reduce one or more undesirable symptoms of the condition ordisorder.

Thus, where CD40 is associated with an undesirable immune response orprocess in vivo, such as autoimmunity, hypersensitivity, inflammation ortransplant rejection, a peptide disclosed herein or a pharmaceuticalcomposition disclosed herein can be administered to a subject having, orat risk of having autoimmunity, hypersensitivity, inflammation ortransplant rejection in order to induce immunosuppression and inhibit orprevent autoimmunity, hypersensitivity, inflammation or transplantrejection in the subject.

Accordingly, in one embodiment, the method of inducing immunosuppressiondisclosed herein can be useful to a subject who is suffering from or isat a risk of developing an inflammatory disease. “Inflammatory disease”means an immune-mediated inflammatory condition, generally characterizedby dysregulated expression of one or more cytokines. Examples ofinflammatory disease include skin inflammatory disorders, inflammatorydisorders of the joints, and inflammatory disorders of thecardiovascular system, autoimmune diseases, lung and airway inflammatorydisorders, intestinal inflammatory disorders. Examples of skininflammatory disorders include dermatitis, for example atopic dermatitisand contact dermatitis, acne vulgaris, and psoriasis. Examples ofinflammatory disorders of the joints include rheumatoid arthritis.Examples of inflammatory disorders of the cardiovascular system arecardiovascular disease and atherosclerosis. Examples of autoimmunediseases include Type 1 diabetes, Graves' disease, Guillain-Barredisease, Lupus, Psoriatic arthritis, and Ulcerative colitis. Examples oflung and airway inflammatory disorders include asthma, cystic fibrosis,COPD, emphysema, and acute respiratory distress syndrome. Examples ofintestinal inflammatory disorders include colitis and inflammatory boweldisease. Other inflammatory disorders include cancer, hay fever,periodontitis, allergies, hypersensitivity, ischemia, depression,systemic diseases, post infection inflammation, amyotrophic lateralsclerosis and bronchitis.

As used herein, the term “autoimmune disease” refers generally to thosediseases characterized by the failure of one or more B- and/or T-cellpopulations, or gene products thereof, to distinguish between self andnon-self antigenic determinants. Autoimmune diseases are oftencharacterized by the infiltration of the target cells with inflammatorylymphoid cells, for example, mononuclear phagocytes, lymphocytes andplasma cells as well as secondary lymphoid follicles. Exemplaryautoimmune diseases include, but are not limited to, organ specificdisorders such as Hashimoto's thyroiditis, primary myxoedemathyrotoxicosis, pernicious anemia, Addison's disease, andinsulin-dependent diabetes mellitus as well as non-organ specificdisorders such as systemic lupus erythematosus (SLE), rheumatoidarthritis (RA), multiple sclerosis, dermatomyositis, scleroderma andpsoriasis. Non-limiting examples of autoimmune disorders treatable witha peptide or pharmaceutical composition of the disclosure includerheumatoid arthritis, lupus (e.g., SLE, lupus nephritis), production ofauto-antibodies which, in the case of antibodies against myelin basicprotein contribute to multiple sclerosis and in the case of antibodiesagainst insulin contribute to diabetes, and Crohn's disease.Non-limiting examples of hypersensitivity treatable with a peptidedisclosed herein or pharmaceutical compositions of the disclosureinclude allergic reactions to antigens, antibiotics, etc.

Non-limiting examples of inflammation treatable with a peptide orpharmaceutical composition of the present disclosure include vascularinflammatory disease (e.g., artherosclerotic lesions, plaque disruptionand thrombus formation), production of inflammatory cytokines (e.g.,LIF, GM-CSF, and IL-6), lung fibrosis and inflammation associated withmultiple sclerosis or a tissue or organ transplant. CD40 activity can beassociated with inflammation caused by viral myocarditis and, as such, apeptide or pharmaceutical composition disclosed herein can be used toinhibit inflammation associated with viral infection.

Additional situations exist in which it may be desired to inhibit animmune response. For example, production of neutralizing antibodiesagainst therapeutic agents, such as anti-insulin antibodies in diabeticsadministered insulin repeatedly, or in subjects that produce anti-virusantibodies (e.g., adenovirus or adeno-associated virus) being treatedwith a gene therapy virus vector, may be inhibited using a peptide orpharmaceutical composition herein. Accordingly, provided herein is amethod of treating a subject suffering from an inflammatory disease, themethod comprising, administering to the subject an effective amount of apharmaceutical composition disclosed herein.

CD40 is also present in various other tissues and cells. For example,CD40 is present in epithelial cells, vascular endothelium and smoothmuscle cells, and CD40 ligand (CD 154) was expressed bythrombin-activated platelets. These findings indicate a role for CD40activity in vascular thrombotic-atheromatic pathophysiology. Therefore,a peptide disclosed herein or a pharmaceutical composition of thepresent disclosure that decreases a CD40 activity can be used to inhibitthrombus formation or artherosclerosis in a subject.

CD40 is also expressed in human renal tubules, thymic epithelia andneural cells. Interestingly, CD40 activity appears to induce apoptosisin neural cells, in contrast to its cell survival role in the immunesystem. Thus, a peptide or pharmaceutical composition herein can be usedto inhibit apoptosis in neural cells. Accordingly, CD40 can be used totreat neural disorders characterized by cell degeneration or undesirableor excessive cell death, such as Parkinson's disease, Alzheimer's,Huntington disease, spinocerebellar ataxias/atrophies, etc.

CD40 is also expressed in carcinomas, such as melanoma, Kaposi'ssarcoma, osteosarcoma and Ewing' sarcoma. CD40 in malignant melanomaappears to be predictive of a negative prognosis. CD40 in human bladdercarcinoma cells inhibits fas-mediated apoptosis. CD40 has been detectedin tumor vasculature in a renal carcinoma mass. Stimulation of CD40 inB-cell lymphomas stimulates growth. CD40 can therefore function as acell survival or growth factor in some tumors, and may promoteangiogenesis. Other data indicate that CD40 may induce cell death intransformed cells.

Thus, a peptide or compositions disclosed herein that decreases a CD40activity associated with tumor survival will be useful in treating cellproliferative disorders (e.g., tumors) in which CD40 functions as a cellsurvival signal or growth promoter, either directly (e.g., in the tumorcell) or indirectly (i.e., through stimulation of angiogenesis within atumor mass). Other biological pathways and physiological conditions thatCD40 participates in are described in Biancone, et al. (Int. J. Mol.Med. 3:343 (1999)), Laman et al. (Dev. Immunol. 6:215 (1998)), Kootenand Bachereau (J. Leukoc. Biol. 67:2 (2000)), Noelle et al. (Ann. NYAcad. Sci. 815:384 (1997)), Noelle (Immunity 4:415 (1996)), Grewal etal. (Curr. Opin. Immunol. 9:491 (1997)); Grewal et al. (Ann. Rev.Immunol. 16:111 (1998); Grewal et al. (Immunol. Rev. 153:85 (1996));Gruss et al. (Leuk. Lymphoma 24(5-6):393 (1997)); van Kooten et al.(Curr. Opin. Immunol. 9:330 (1997)). Such pathways as well as othersknown in the art are amenable to modulation using the peptides disclosedherein or compositions comprising said peptides disclosed herein, as arethe physiological conditions associated with CD40 activity describedtherein or otherwise known in the art.

Prevention or Treatment of a Cancer

The peptides or compositions disclosed can find use in treatment of aneoplastic disease. In one aspect, provided herein is a method oftreating a subject suffering from or is at risk of developing aneoplastic disease characterized by a neoplastic cell that expressesCD40, the method comprising administering to the subject an effectiveamount of peptides disclosed herein or a composition comprising thepeptides disclosed herein. CD40 has been shown to be expressed not onlyon immune cells, but also on tumor cells, including B-cell malignanciesand solid tumors.

As used herein, the term “neoplastic cells that expresses CD40,” refersto a cell (e.g., an immune cell such as B-cell, T-cell, or an non-immunecell, such as epithelial cell, endothelial cell, smooth muscle cell)that expresses a CD40 protein on its cell surface and exhibits abnormalcell growth, cell division and proliferation not undergoing apoptosis,or both, under inappropriate conditions. For example, a “neoplasticcell” may undergo cell division when a corresponding non-neoplastic celldoes not undergo cell division, or, alternatively, a “neoplastic cell”may not respond to normal cellcycle checkpoint controls. Accordingly aneoplastic cell is one that exhibits abnormal proliferation of cells.The growth of neoplastic cells exceeds that of normal tissue around itand it is not coordinated with that of the normal tissue around itresulting in neoplasms. Neoplasms may be benign (e.g., benign tumor andatypical hyperplasia), pre-malignant (e.g., carcinoma in situ andpre-cancer) or malignant (e.g., cancer). The term “cancer” refers to anyof the various malignant neoplasms characterized by the proliferation ofcells that have the capability to invade surrounding tissue and/ormetastasize to new colonization sites, including but not limited toleukemias, lymphomas, carcinomas, melanomas, sarcomas, germ cell tumorsand blastomas. Exemplary cancers include cancers of the brain, bladder,breast, cervix, colon, head and neck, kidney, lung, non-small cell lung,mesothelioma, ovary, prostate, stomach and uterus, leukemia andmedulloblastoma.

Neoplastic tissues can originate from any cell type or tissue found in amammal, including, but not limited to hepatic, skin, breast, prostate,neural, optic, intestinal, cardiac, vasculature, lymph, spleen, renal,bladder, lung, muscle, connective, tissue, pancreatic, pituitary,endocrine, reproductive organs, bone, and blood. The neoplastic tissuefor analysis may include any type of solid tumor or hematologicalcancer. In some embodiments, the neoplastic tissue is a breast cancertissue. In other embodiments, the neoplastic tissue is a breast tissuewith atypical hyperplasia.

The term “leukemia” refers to broadly progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia diseases include, for example, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, acutegranulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas include, for example, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypemephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

The term “sarcoma” generally refers to a tumor which arises fromtransformed cells of mesenchymal origin. Sarcomas are malignant tumorsof the connective tissue and are generally composed of closely packedcells embedded in a fibrillar or homogeneous substance. Sarcomasinclude, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adiposesarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma,Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing'ssarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphomas (e.g.,Non-Hodgkin Lymphoma), immunoblastic sarcoma of T-cells, Jensen'ssarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma,leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma,reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovialsarcoma, and telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas include, forexample, acral-lentiginous melanoma, amelanotic melanoma, benignjuvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passeymelanoma, juvenile melanoma, lentigo maligna melanoma, malignantmelanoma, nodular melanoma subungal melanoma, and superficial spreadingmelanoma.

The Examples herein show that the CD40 binding peptides disclosed hereincan bind CD40 expressed on B-cell and inhibit, for example, B-cellproliferation and B-cell activation. The peptides of the presentdisclosure can find use, for example, in the treatment of B-celllymphomas, prevention of EBV-induced B-cell lymphoma that can occurafter transplantation or in other instances of immunosuppression, suchas AIDS, and which present a significant risk in such patientpopulations. The methods disclosed herein can be useful in prevention ofimmunoblastic B-cell lymphomas that frequently arise inimmunocompromised individuals. In such preventative methods, a mammal atrisk of developing an immunoblastic B-cell lymphoma is administered aneffective amount of peptides disclosed herein. The peptides can beadministered for as long as the state of immunocompromise that placesthe individual at risk exists.

The peptides of the present disclosure or compositions comprising thepeptides can be useful for treatment of cancer characterized by“neoplastic B-cell growth” is intended any disease or condition(including pre-malignant conditions) involving uncontrolled growth ofcells of B-cell lineage. Such diseases and conditions include, but arenot limited to, acute lymphoblastic leukemia (ALL), acute myelogenousleukemia (AML), chronic myelogenous leukemia (CIVIL), chroniclymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), smalllymphocytic leukemia (SLL), diffuse small lymphocytic leukemia (DSLL),diffuse large B-cell lymphoma (DLBCL), hairy cell leukemia,non-Hodgkin's lymphomas, Hodgkin's disease, Epstein-Barr Virus (EBV)induced lymphomas, myelomas such as multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, mucosal associated lymphoidtissue lymphoma, monocytoid B cell lymphoma, splenic lymphoma,lymphomatoid granulomatosis, intravascular lymphomatosis, immunoblasticlymphomas, AIDS-related lymphomas, and the like.

In another aspect, the compositions and methods used to preventoccurrence (or reoccurrence) or treatment of neoplastic diseasecharacterized by other types of malignant cells that express CD40 inindividuals at risk for such disease. Individuals that are considered atrisk in these instances include those with family history or othergenetic characteristics indicating predisposition to cancers in whichthe neoplastic cells express CD40, and individuals that developdrug-resistant neoplastic disease as a result of chemotherapy, in whichthe drug-resistant neoplastic cells express CD40.

Individuals afflicted with disease characterized by neoplastic cellsthat express CD40 may also be treated according to the methods disclosedherein. The term treatment, as it is generally understood in the art,refers to initiation of therapy after clinical symptoms or signs ofdisease have been observed. The methods may be used in conjunction withother therapies appropriate for afflicted individuals, includingchemotherapy, radiation therapy, and immunotherapy.

The “effective amount” to be administered will be governed by suchconsiderations, and refers to the minimum amount necessary toameliorate, treat, or stabilize, the cancer; to increase the time untilprogression (duration of progression free survival) or to treat orprevent the occurrence or recurrence of a tumor, a dormant tumor, or amicrometastases. The peptides, disclosed herein, can be optionallyformulated with one or more additional therapeutic agents currently usedto prevent or treat cancer or a risk of developing a cancer. Theeffective amount of such other agents depends on the amount of peptidespresent in the formulation, the type of disorder or treatment, and otherfactors such age, weight etc. These are generally used in the samedosages and with administration routes as used herein before or aboutfrom 1 to 99% of the heretofore employed dosages.

The efficacy of the treatment methods for cancer, comprisingadministering the peptides disclosed herein or pharmaceuticalcompositions of the present disclosure can be measured by variousendpoints commonly used in evaluating cancer treatments, including butnot limited to, tumor regression, tumor weight or size shrinkage, timeto progression, duration of survival, progression free survival, overallresponse rate, duration of response, and quality of life. The peptidesdisclosed herein can require unique measures and definitions of clinicalresponses to drugs. In the case of cancers, the therapeuticallyeffective amount of the peptides disclosed herein or compositionscomprising the same can reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the disorder. To the extent the peptidesdisclosed herein, act to prevent growth and/or kill existing cancercells; it can be cytostatic and/or cytotoxic. For cancer therapy,efficacy in vivo can, for example, be measured by assessing the durationof survival, duration of progression free survival (PFS), the responserates (RR), duration of response, and/or quality of life.

In other embodiments, described herein are methods for increasingprogression free survival of a human subject susceptible to or diagnosedwith a cancer, for example, skin cutaneous melanoma. Time to diseaseprogression is defined as the time from administration of the drug untildisease progression or death. In a preferred embodiment, the combinationtreatment of the disclosure using a peptide or compositions disclosedherein, and one or more chemotherapeutic agents may significantlyincrease progression free survival by at least about 1 month, 1.2months, 2 months, 2.4 months, 2.9 months, 3.5 months, such as by about 1to about 5 months, when compared to a treatment with chemotherapy alone.In another embodiment, the methods described herein may significantlyincrease response rates in a group of human subjects susceptible to ordiagnosed with a cancer that are treated with various therapeutics.Response rate is defined as the percentage of treated subjects whoresponded to the treatment. In one embodiment, the combination treatmentdescribed herein using a peptide or compositions disclosed herein, andone or more chemotherapeutic agents significantly increases responserate in the treated subject group compared to the group treated withchemotherapy alone.

For example, in some embodiments, the methods described herein compriseadministering an effective amount of the peptides of the instantdisclosure or compositions comprising said peptides, to a subject inorder to alleviate a symptom of a cancer. As used herein, “alleviating asymptom of a cancer” is ameliorating or reducing any condition orsymptom associated with the cancer. As compared with an equivalentuntreated control, such reduction or degree of prevention is at least5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by anystandard technique. Ideally, the cancer is completely cleared asdetected by any standard method known in the art, in which case thecancer is considered to have been treated. A patient who is beingtreated for a cancer is one who a medical practitioner has diagnosed ashaving such a condition. Diagnosis can be by any suitable means.Diagnosis and monitoring can involve, for example, detecting the levelof cancer cells in a biological sample (for example, a tissue or lymphnode biopsy, blood test, or urine test), detecting the level of asurrogate marker of the cancer in a biological sample, detectingsymptoms associated with the specific cancer, or detecting immune cellsinvolved in the immune response typical of such a cancer.

The treatment and/or prevention of cancer includes, but is not limitedto, alleviating symptoms associated with cancer, the inhibition of theprogression of cancer, the promotion of the regression of cancer, thepromotion of the immune response, inhibition of tumor growth, inhibitionof tumor size, inhibition of metastasis, inhibition of cancer cellgrowth, inhibition of cancer cell proliferation, or cause cancer celldeath.

Transplant Immunomudulation

Described herein are compositions, systems, and methods for inducinggraft tolerance. In particular, the present disclosure relates toadministering a tolerizing vaccine or a preparatory regimen before,during, and/or after administration of donor transplant cells,tissue(s), or organ(s). The tolerizing vaccine or preparatory regimencan induce tolerance to the allograft or xenograft in the graftrecipient.

An organ, tissue, or cell can be differentiated from stem cells, grownde novo, or isolated from an animal (e.g., a human or non-human animal)and can be transplanted into a recipient in need of a transplant fromthe same species (an allotransplant) or a different species (axenotransplant). The donor of the organ, tissue, or cell can be referredto herein as a transplant donor. The transplanted organ, tissue, or cellcan be referred to herein as a transplant or a graft.

The donor of transplant or graft can be any animal, including human andnon-human animals. In some cases the donor is a human. In some cases,the transplant donor is a non-human animal. The transplant donor (e.g.,a non-human animal donor) can be genetically modified, for example, toreduce or eliminate the likelihood that the transplant or graft can berecognized by the recipient's immune system, or to reduce the immuneresponse by the recipient's immune system upon recognizing thetransplant or graft.

The donor of a transplant or graft can be a living donor or a cadavericdonor. In some cases, the transplant donor is a living donor. In somecases, the transplant donor is a cadaveric donor. The cadaveric donormay be a brain dead, heart beating donor (BDD). Alternatively, thecadaveric donor may be a non-heart beating donor (NHBD). Whether thetransplant donor is a living donor or a cadaveric donor (e.g., a BDD orNHBD), the donor can be from any animal, for example, a human ornon-human animal.

The donor of a transplant or graft can be at any age or stage ofdevelopment. For example, the transplant donor can be a fetal,perinatal, neonatal, pre-weaning, post-weaning, juvenile, young adult,or adult human or animal.

Transplants or grafts can be used to treat diseases or disorders inrecipients in need thereof. Suitable diseases that can be treated areany in which an organ, tissue, or cell of a recipient is defective orinjured, and the recipient can be treated by transplantation of anorgan, tissue, or cell (e.g., a kidney, heart, lung, liver, vein, skin,endocrine pancreas, pancreatic islet cell, or a combination thereof). Insome cases, the transplant comprises a kidney, liver, heart, lung,pancreas, endocrine pancreas, islet cell, small bowel, bone marrow,hematopoietic stem cell, embryonic or induced pluripotent stemcell-derived cells such as islet beta cells or hepatocytes, embryonic orinduced pluripotent stem cell-derived islet, embryonic or inducedpluripotent stem cell-derived hepatocyte, or a combination thereof.

Inhibiting Immune Response to a Donor Cell or Tissue Transplant.

In some embodiments, methods for inducing immunosuppression can beuseful to prevent graft rejection in a subject who has undergone, isundergoing or will be undergoing a cell, organ or tissue transplant.

The peptides and compositions disclosed herein can find use inpreventing transplant rejection by inhibiting a CD40 mediated immuneresponse in a recipient, towards an antigen on a transplanted material(e.g., cells, tissues, an/or organs). In one aspect provided herein is amethod of inhibiting an immune response to a donor cell or tissuetransplantation in a recipient, the method comprising administering tothe recipient a peptide or compositions disclosed herein. Examples oftransplant rejection (acute or chronic) treatable with a peptidedisclosed herein include blood vessels, kidney, liver, heart, lung,pancreas and skin. Transplantation includes grafting of tissues or organfrom the body of an individual to a different place within the sameindividual, or a different individual. Transplantation also involvesgrafting of tissues or organs from one area of the body to another.Transplantation of tissues or organs between genetically dissimilaranimals of the same species is termed as allogeneic transplantation.Transplantation of animal organs into humans is termed xenotransplants.

Transplant/graft rejection can involve recognition of donor-specificantigens, for example, recognition of donor-specific antigens presentedto T cells by host antigen presenting cells (indirect) or donor antigenpresenting cells (direct). T cell activation in response todonor-specific antigens can lead to a pro-inflammatory response andtransplant rejection. Transplant rejection (e.g., T cell-mediatedtransplant rejection) can be prevented by chronic immunosuppression withone or more immunomodulatory molecules. However, immunosuppression iscostly and associated with the risk of serious side effects.

In some embodiments, a method described herein to preventtransplantation rejection or prolong the time to transplantationrejection without or with minimal immunosuppressive drug use (e.g., oneor more immunomodulatory molecules) involves using a geneticallymodified animal as a cell, organ, or tissue donor. The cells, organs,and/or tissues of the altered donor animal, e.g., a donor non-humananimal, can be harvested and used in allografts or xenografts.Alternatively, a cell, organ, or tissue can be extracted from an animal,and used to generate a genetically-altered cell, organ or tissue. Insome cases, primary cells can be extracted from an animal, and used tomake genetically altered cells. In some cases, a cell, organ, or tissuederived from an animal (e.g., a cell line) can be used to create agenetically altered cell, organ, or tissue.

Transplant rejection can also be reduced or eliminated by inducingtolerance to a transplant or graft using a tolerizing vaccine orpreparatory regimen. A tolerizing vaccine or preparatory regimen of thedisclosure can be used to prevent transplant rejection or delayrejection, for example, by reducing a pro-inflammatory immune responseto the transplant, and/or enhancing a tolerance-promoting immuneresponse. In some cases, a tolerizing vaccine or preparatory regimen ofthe disclosure can circumvent the need for long-term immunosuppressionof the recipient.

An effective amount, which is determined by these considerations, is theminimum amount necessary to inhibit an immune response in a recipientthat would result in rejection of the transplant, but as much asnecessary to achieve a longer transplant survival time. Such amount ispreferably below the amount that is toxic to the recipient or rendersthe recipient significantly more susceptible to infections. The amountof peptides or compositions disclosed herein used for inhibiting immuneresponse directed to a transplant can be lower than the amount of animmunosuppressive agent normally required for transplanted grafts anddepends on the individual circumstances surrounding the transplant. Atreatment with the peptides or compositions disclosed herein can alsolessen or prevent the side effects frequently observed in transplantrecipients who undergo immune suppressive therapy,

The present disclosure therefore provides a method for inhibitingtransplant rejection and a method to increase transplant survival. Inone aspect, the peptides or compositions disclosed herein canadministered as a part of a transplantation protocol. Accordingly,provided herein is a method for transplantation of a cell, organ, ortissue from a mammalian donor in a recipient, the method comprisingadministering an effective amount of the peptides or pharmaceuticalcompositions disclosed herein. In some embodiments, the method oftransplantation further comprises transplanting the donor cell tissue ororgan in a recipient.

An organ, tissue, or cell isolated from an animal (e.g., a human ornon-human animal) can be transplanted into a recipient in need of atransplant from the same species (an allotransplant) or a differentspecies (a xenotransplant). Transplants or grafts can be used to treatdiseases or disorders in a recipient in need thereof. Suitable diseasesthat can be treated are any in which an organ, tissue, or cell of arecipient is defective or injured, (e.g., a kidney, heart, lung, liver,vein, skin, pancreatic islet cell, or a combination thereof) and therecipient can be treated by transplantation of an organ, tissue, orcell. In some cases, the transplant comprises a kidney, liver, heart,lung, pancreas, islet cell, small bowel, bone marrow, hematopoietic stemcell, embryonic or induced pluripotent stem cell-derived cells,embryonic or induced pluripotent stem cell-derived islet, embryonic orinduced pluripotent stem cell-derived hepatocyte, or a combinationthereof.

The methods herein can comprise administering one or more dose of apeptide or compositions disclosed herein to a recipient before, after,and/or during transplant of donor cells, organs, and/or tissues toinhibit donor-specific immune response in a recipient. In some cases, afirst dose of a peptide or compositions disclosed herein can be given onor on about day −100, day −90, day −80, day −70, day −60, day −50, day−40, day −30, day −20, day −15, day −14, day −13, day −12, day −11, day−10, day −9, day −8, day −7, day −6, day −5, day −4, day −3, day −2 orday −1, relative to transplant of donor cells, organs, and/or tissues onday 0. In some cases, a first dose of a peptide or compositionsdisclosed herein can be given on or on about day −100 to −50; −50 to−40; −40 to −30; −30 to −20; −20 to −10; −10 to −5; −7 to −1, relativeto transplant of donor cells, organs, and/or tissues on day 0. Forexample, a first dose of a peptide or compositions disclosed herein canbe given 8 days (e.g., day −8) before transplant of donor cells, organs,and/or tissues. In some embodiments, a peptide or compositions of thepresent disclosure can be administered on the same day (e.g., day 0) astransplant of donor cells, organs, and/or tissues. In some cases, apeptide or a composition disclosed herein can be administered on or onabout day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50,day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10,day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1,relative to transplant of donor cells, organs, and/or tissues on day 0.For example, a peptide or compositions disclosed herein can beadministered on or on about day 400 to 350; 350 to 300; 300 to 250; 250to 200; 200 to 150; 150 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20;20 to 10; 10 to 5; 7 to 1, relative to transplant of donor cells,organs, and/or tissues on day 0. For example, a peptide or compositionsdisclosed herein can be administered on 1 day after (e.g., day 1)transplant of donor cells, organs, and/or tissues. In some embodiments,a peptide or compositions disclosed herein can be administered of day 14after transplant of donor cells, organs, and/or tissues. In someembodiments, a peptide or compositions disclosed herein can beadministered for a lifetime of the recipient.

A recipient (e.g., a human or a non-human animal) can requireadministration with a peptide or composition disclosed herein for atleast or at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 900, or 1,000 days after transplantation,e.g., for at least or at least about 1 to 5; 5 to 10; 10 to 20; 20 to30; 30 to 60; 60 to 100; 100 to 200; 200 to 300; 300 to 400; 400 to 500;500 to 600; 600 to 700; 700 to 800; 800 to 900; 900 to 1,000 days. Arecipient (e.g., a human or a non-human animal) can requireadministration of a peptide or composition disclosed herein for at leastor at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, or 36 months after transplantation, e.g., for at least or at leastabout 1 to 2; 2 to 3; 3 to 6; 6 to 9; 9 to 12; 12 to 18; 18 to 24; 24 to30; 30 to 36 months after transplantation. A recipient (e.g., a human ora non-human animal) can require administration of a peptide or acomposition disclosed herein for at least or at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years after transplantation, e.g.for at least or at least about 1 to 2; 2 to 3; 3 to 4; 4 to 5; 1 to 5; 5to 10; 10 to 15; 15 to 20; 20 to 25; 25 to 30 years aftertransplantation. In some cases, a recipient (e.g., a human or anon-human animal) can require administration of a peptide or acomposition disclosed herein for up to the lifetime of the recipient.

A recipient (e.g., a human or a non-human animal) can requireadministration for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, or 36 months after transplantation e.g.,for at least or at least about 1 to 2; 2 to 3; 3 to 6; 6 to 9; 9 to 12;12 to 18; 18 to 24; 24 to 30; 30 to 36 months after transplantation. Arecipient (e.g., a human or a non-human animal) can requireadministration of a peptide or a composition disclosed herein for atleast or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30years after transplantation, e.g. for at least or at least about 1 to 2;2 to 3; 3 to 4; 4 to 5; 1 to 5; 5 to 10; 10 to 15; 15 to 20; 20 to 25;25 to 30 years after transplantation.

The transplantation method of the present disclosure can comprisemultiple doses of a peptide or a composition disclosed herein before,and/or during and/or after transplantation of a graft cell, tissue, ororgan. The multiple doses can be referred to as comprising an initialdose and one or more booster doses. Typically, the initial dose occursprior to or concurrently with the transplant of the graft cell tissue ororgan. The booster dose(s), when administered, occur after the initialdose. In some embodiments, a booster dose is administered to achieve andmaintain a particular serum/blood trough level of the peptides disclosedherein.

Depending upon when the initial dose of the peptide or compositiondisclosed herein is administered, one or more booster doses can beadministered before, and/or concurrently with, and/or after transplantof the graft cell, tissue, or organ.

Subsequent (e.g., booster) dose(s) of the peptide or compositionsdisclosed herein can be administered in any interval of time following apreceding dose (e.g., an initial dose). For example, the subsequent dosecan be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55days, 56 days, 57 days, 58 days, 59 days, 60 days, 90 days, 120 days,150 days, or 180 days after the preceding dose. Depending upon when theinitial dose is administered subsequent (booster) dose(s) can beadministered before, concurrently with, or after transplantation of thegraft cell, tissue, or organ. In some cases, the methods of the presentdisclosure comprise at least one dose of the peptide or compositionsdisclosed herein prior to transplantation. In some cases, the methods ofthe present disclosure comprises at least two doses of a peptide orcompositions disclosed herein prior to transplantation (e.g., an initialdose and a booster dose). In some cases, the methods herein comprises atleast three doses of the peptide or compositions disclosed herein priorto transplantation (e.g., an initial dose and two booster doses). Insome cases, the preparatory regimen comprises an initial dose of thepeptide or compositions disclosed herein prior to transplantation and atleast one dose of booster concurrently with or after transplantation ofthe graft cell, tissue, or organ.

In some cases, two doses of a peptide or compositions thereof can beadministered. For example, the first dose can be administered on day −8relative to transplant of donor cells, organs, and/or tissues on day 0.For example, the second dose can be administered on day −1 relative totransplant of donor cells, organs, and/or tissues on day 0. For example,the first dose can be administered on day −8, −9, −10, −11, −12, −13, or−14 relative to transplant of donor cells, organs, and/or tissues on day0. For example, the second dose can be administered on day −1, −2, −3,−4, −5, or −6 relative to transplant of donor cells, organs, and/ortissues on day 0. In some cases, a second dose (e.g., a booster dose)can be administered on day 100, day 90, day 80, day 70, day 60, day 50,day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day 23,day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day 14,day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day4, day 3, day 2 or day 1, relative to transplant of donor cells, organs,and/or tissues on day 0. For example, the second dose (e.g., a boosterdose) can be administered on 1 day after (e.g., day 1) transplant ofdonor cells, organs, and/or tissues.

In some cases, a third dose of a peptide or compositions disclosedherein (e.g., a booster dose) can be administered on day 300, day 200,day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29,day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20,day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11,day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1,relative to transplant of donor cells, organs, and/or tissues on day 0.For example, the peptide or compositions disclosed herein can beadministered on or on about day 300 to 200; 200 to 100; 100 to 50; 50 to40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative totransplant of donor cells, organs, and/or tissues on day 0.

In some cases, a fourth dose of a peptide or compositions disclosedherein (e.g., a booster vaccine) can be administered on day 600, day500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5,day 4, day 3, day 2 or day 1, relative to transplant of donor cells,organs, and/or tissues on day 0. For example, the peptide orcompositions disclosed herein can be administered on or on about day 600to 500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative totransplant of donor cells, organs, and/or tissues on day 0.

In some cases, a fifth dose of a peptide or compositions disclosedherein (e.g., a booster dose) can be administered on day 1,000, day 900,day 800, day 700, day 600, day 500, day 400, day 300, day 200, 100, day90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative totransplant of donor cells, organs, and/or tissues on day 0. For example,the peptide or compositions disclosed herein can be administered on oron about day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to500; 500 to 400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative totransplant of donor cells, organs, and/or tissues on day 0. In somecases, a second dose of a booster is not required. In some cases, asecond dose of a booster is given concomitantly on day 0 with transplantdonor cells, organs, and/or tissues.

Booster doses of a peptide or compositions disclosed herein can be lowerdose than the initial or preceding dose of the peptide or compositionsdisclosed herein. For example, a booster or subsequent dose can beabout: 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,or 75% lower than the initial or preceding dose.

Preparatory Regimen/Tolerizing Agent

In some embodiments, the transplantation method comprises administeringa tolerizing agent in the subject. The term “tolerizing agent” or a“toleragen” as used herein means any donor antigen (such as a protein,nucleic acid, carbohydrate, lipid, or combination of any thereof) thatmediates host unresponsiveness. By way of example, a tolerizing agentworks by inducing the tolerized host not to produce antibodies orcell-mediated immune responses specific for the toleragen. Additionaldiscussion of tolerizing may be found, for instance, in PCT publicationWO 2006/052668, which is incorporated herein in its entirety. In someembodiments, the tolerizing agent can be a tolerizing vaccine comprisinginactivated or apoptotic donor cells that upon intravenous injection canresult in tolerance to transplanted donor cells or tissues orcompositions comprising said cells with similar marks. In someembodiments, the tolerizing agent can be an anti-CD40 agent (e.g., agentcomprising one or more amino acid sequences listed in any one of SEQ ID1-6 and 8-17). In some embodiments, the tolerizing agent can bemammalian leukocytes fixed with a crosslinking agent. In some cases, thetolerizing agent can be mammalian leukocytes fixed with a crosslinkingagent, administered together with an anti-CD40 binding agent (e.g.,agent comprising one or more amino acid sequences listed in any one ofSEQ ID 1-6 and 8-17). In some cases, the tolerizing agent can bemammalian leukocytes fixed with a crosslinking agent, surface-conjugatedwith an anti-CD40 binding agent (e.g., agent comprising one or moreamino acid sequences listed in any one of SEQ ID 1-6 and 8-17).

In some embodiments, the preparatory regimen can be inactivated orapoptotic donor cells that upon intravenous injection can result intolerance to transplanted donor cells or tissues or compositionscomprising said cells with similar marks. In some embodiments, thepreparatory regimen can be an anti-CD40 agent (e.g., agent comprisingone or more amino acid sequences listed in any one of SEQ ID 1-6 and8-17). In some embodiments, the preparatory regimen can be mammalianleukocytes fixed with a crosslinking agent. In some cases, thepreparatory regimen can be mammalian leukocytes fixed with acrosslinking agent, administered together with an anti-CD40 bindingagent (e.g., agent comprising one or more amino acid sequences listed inany one of SEQ ID 1-6 and 8-17). In some cases, the preparatory regimencan be fixed mammalian leukocytes surface-conjugated with an anti-CD40binding agent (e.g., agent comprising one or more amino acid sequenceslisted in any one of SEQ ID 1-6 and 8-17).

Robust tolerance to allografts can be achieved without requiring samedonor bone marrow or hematopoietic stem cell transplantation with theassociated intense conditioning therapy by employing a negative vaccinestrategy. In an exemplary embodiment, a tolerizing vaccine orpreparatory regimen of the disclosure can comprise administering ananti-CD40 agent or an anti-CD40 ligand agent, e.g., any one or more ofthe peptides comprising an amino acid sequence with at least 80%, atleast 85%, at least 90% or at least 95% sequence identity to thesequences set forth in any one of SEQ ID NOs. 1-6 and 8-17. In addition,the tolerizing vaccine or preparatory regimen can further compriseimmunosuppression agents such as (i) an mTOR inhibitor, (ii) ananti-tumor necrosis factor agent or an anti-tumor necrosis factorreceptor agent, or (iii) an anti-interleukin 6 agent or ananti-interleukin 6 receptor agent.

In another embodiment, a tolerizing vaccine or preparatory regimen ofthe disclosure can comprise administering apoptotic cells (e.g.,apoptotic donor leukocytes) to a recipient conjugated to an anti-CD40agent or an anti-CD40 ligand agent, e.g., any one or more of thepeptides comprising an amino acid sequence with at least 80%, at least85%, at least 90% or at least 95% sequence identity to the sequences setforth in any one of SEQ ID NOs. 1-6 and 8-17. The short-termadministration of these agents along with apoptotic donor leukocytes asdisclosed herein can promote long-term tolerance to a transplanted cell,organ, or tissue despite not administering long term maintenanceimmunosuppression to the recipient.

Sources of Cells for a Tolerizing Vaccine/Regimen or Preparatory Regimen

Cells for the preparation of a tolerizing vaccine or preparatory regimencan be obtained from any source, including animals, cells lines, and/orlab-generated cells. For example, the cells can be obtained from a humanor non-human animal. In another example, the cells can be from a cellline (e.g., a human or non-human cell line). In some cases, the cellsare human cells. In other cases, the cells are non-human cells. In somecases, the cells are of a non-human primate. In some cases, the cellsare of a member of the Laurasiatheria superorder. In some cases, thecells are of an ungulate, for instance a camelid or a pig. In somecases, the cells are of a pig. In some cases, the cells are from thesame species as the transplant donor. In some cases, the cells are fromthe same species as the transplant recipient. In some cases, the cellsare from the same species as the transplant donor and the transplantrecipient. In some cases, the cells are from a different species thanthe transplant donor. In some cases, the cells are from a differentspecies than the transplant recipient. In some cases, the cells are froma different species than the transplant donor and the transplantrecipient.

Cells for the preparation of a tolerizing vaccine or preparatory regimencan be obtained from living donors or cadaveric donors. In some cases,the donor is a living donor. In some cases, the donor is a cadavericdonor. The cadaveric donor may be, for example, a brain dead, heartbeating donor (BDD). The cadaveric donor may be, for example, anon-heart beating donor (NHBD). Whether the donor is a living donor or acadaveric donor (e.g., a BDD or NHBD), the donor can be from any animal,for example, a human or non-human animal. In some cases, cells for thepreparation of a tolerizing vaccine can be from the same donor as agraft or transplant. In some cases, cells for the preparation of atolerizing vaccine can be from a different donor than the graft ortransplant.

Cells for the preparation of a tolerizing vaccine or preparatory regimencan be obtained from a donor animal of any age or stage of development.For example, the donor animal can be a fetal, perinatal, neonatal,pre-weaning, post-weaning, juvenile, young adult, or adult animal. Insome cases, non-human animals can be past weaning age. For example,donor animals can be at least or at least about six months old. In somecases, donor animals can be at least or at least about 18 months old. Insome cases, cells for the preparation of a tolerizing vaccine orpreparatory regimen can be obtained (for example, differentiated) fromstem cells (e.g., embryonic stem cells, induced pluripotent stem cells,and/or mesenchymal stem cells).

The cells used to make a tolerizing vaccine or preparatory regimen caninclude one or more cells from tissues, organs, or bodily fluids. Forexample, the cells can be from tissues, organs, or bodily fluidsincluding, but not limited to: brain, lung, liver, heart, spleen,pancreas, small intestine, large intestine, skeletal muscle, smoothmuscle, skin, bones, adipose tissues, hairs, thyroid, trachea, gallbladder, kidney, ureter, bladder, aorta, vein, esophagus, diaphragm,stomach, rectum, adrenal glands, bronchi, ears, eyes, retina, genitals,hypothalamus, larynx, nose, tongue, spinal cord, or ureters, uterus,ovary, testis, blood, spinal fluid, lymph fluid, or a combinationthereof.

The cells used to make a tolerizing vaccine or preparatory regimen caninclude one or more types of cells. For example, the cells can include,but are not limited to: trichocytes, keratinocytes, gonadotropes,corticotropes, thyrotropes, somatotropes, lactotrophs, chromaffin cells,parafollicular cells, glomus cells melanocytes, nevus cells, Merkelcells, odontoblasts, cementoblasts corneal keratocytes, retina Mullercells, retinal pigment epithelium cells, neurons, glias (e.g.,oligodendrocyte astrocytes), ependymocytes, pinealocytes, pneumocytes(e.g., type I pneumocytes, and type II pneumocytes), clara cells, gobletcells, G cells, D cells, ECL cells, gastric chief cells, parietal cells,foveolar cells, K cells, D cells, I cells, goblet cells, paneth cells,enterocytes, microfold cells, hepatocytes, hepatic stellate cells (e.g.,Kupffer cells from mesoderm), cholecystocytes, centroacinar cells,pancreatic stellate cells, pancreatic α cells, pancreatic β cells,pancreatic δ cells, pancreatic F cells (e.g., PP cells), pancreatic ccells, thyroid (e.g., follicular cells), parathyroid (e.g., parathyroidchief cells), oxyphil cells, urothelial cells, osteoblasts, osteocytes,chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts,myocytes, myosatellite cells, tendon cells, cardiac muscle cells,lipoblasts, adipocytes, interstitial cells of cajal, angioblasts,endothelial cells, mesangial cells (e.g., intraglomerular mesangialcells and extraglomerular mesangial cells), juxtaglomerular cells,macula densa cells, stromal cells, interstitial cells, telocytes simpleepithelial cells, podocytes, kidney proximal tubule brush border cells,sertoli cells, leydig cells, granulosa cells, peg cells, germ cells,spermatozoon ovums, lymphocytes, myeloid cells, endothelial progenitorcells, endothelial stem cells, angioblasts, mesoangioblasts, pericytemural cells, mesenchymal stromal cells, or splenocytes (e.g., Tlymphocytes, B lymphocytes, dendritic cells, microphages, leukocytes).In some cases, the cells used to make a tolerizing vaccine orpreparatory regimen comprise a cell type that expresses MHC class II. Insome cases, the cells used to make a tolerizing vaccine or preparatoryregimen comprise a cell type that does not expresses MHC class II.

A tolerizing vaccine or preparatory regimen can comprise leukocytes.Leukocytes can include, for example, neutrophils, eosinophils,basophils, lymphocytes, monocytes, or a combination thereof. Lymphocytescan include, for example, B lymphocytes (B cells), T lymphocytes (Tcells), natural killer (NK) cells, or a combination thereof.

Leukocytes in a tolerizing vaccine or preparatory regimen can beobtained from any source, including, for example, a donor, a cell line,or a differentiated stem cell. Leukocytes obtained from a donor caninclude leukocytes obtained from a spleen (e.g., splenocytes, splenic Bcells); a liver; peripheral blood (including peripheral blood B cells);a lymph node; a thymus; bone marrow; or any other organ, tissue, orbodily fluid; or any combination thereof. In some cases, the tolerizingvaccine or preparatory regimen comprises splenic B cells, peripheralblood B cells, or a combination thereof. In some cases, the tolerizingvaccine or preparatory regimen comprises cells mobilized from the bonemarrow to peripheral blood with a mobilization agent, e.g., cellsmobilized with granulocyte colony-stimulating factor (G-CSF),granulocyte macrophage colony-stimulating factor (GM-CSF), mozobil, or acombination thereof. The leukocytes in the tolerizing vaccine orpreparatory regimen that are obtained from a donor can comprise primarycells, cells expanded ex vivo, or a combination thereof.

Genetically Modified Cells for a Tolerizing Vaccine or PreparatoryRegimen

A donor of cells used in the preparation of a tolerizing vaccine orpreparatory regimen can be genetically modified. Alternatively, oradditionally, cells obtained from a donor can be genetically modified exvivo. In some cases, cell lines are genetically modified to producecells for use in a tolerizing vaccine or preparatory regimen. Thegenetically modified donors and/or cells can be produced using anymethod known in the art, including those described herein. Regardless ofwhether the genetically modified cells are isolated from a geneticallymodified animal, produced in culture, or a combination thereof, thegenetically modified cells can be of any animal species, including humanand non-human animals.

Genetically modified cells used in a tolerizing vaccine or preparatoryregimen can comprise one or more genetic modifications that reduce oreliminate expression or a gene or gene product (e.g., a protein). Thegenetic modification(s) can be modifications to the gene whoseexpression is reduced or eliminated. Such genes can be referred to asdisrupted genes. The genetic modification(s) can also be to areas of thegenome separate from the gene whose expression is reduced or eliminated(for example, modification to a promoter, enhancer, silencer,transcription factor, etc.). The genetically modified cells used in thetolerizing vaccine or preparatory regimen can comprise, for example, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20,or more genes whose expression is reduced or eliminated by geneticmodification.

Non-limiting examples of genes whose expression can be reduced oreliminated by genetic modification in the cells used in a tolerizingvaccine or preparatory regimen include, but are not limited to: alpha1,3 galactosyltransferase (GGTA1); putative cytidinemonophosphatase-N-acetylneuraminic acid hydroxylase-like protein (CMAH);β1,4N-acetylgalactosaminyltransferase (B4GALNT2); a component of a majorhistocompatibility complex (MHC) I-specific enhanceosome (e.g., aNOD-like receptor family CARD domain containing 5 (NLRC5)); atransporter of an MHC I-binding peptide (e.g., transporter associatedwith antigen processing 1 (TAP1)); complement component 3 (C3); a CXCchemokine receptor 3 ligand (CXCL3); a CXC motif chemokine ligand10(CXCL10) gene; MHC II transactivator (MHCIITA); a MHC class Ipolypeptide-related sequence A (MICA) gene; a MEW class Ipolypeptide-related sequence B (MICB) gene; a natural killer (NK) group2D ligand (NKG2DL); a tumor necrosis factor receptor (TNF-R); a pigendogenous retrovirus (PERV); B2M, PD-1, PD-L1 or any combinationthereof.

In some cases, the genetically modified cells used in a tolerizingvaccine can comprise disruptions in one or more genes comprising GGTA1,CMAH, B4GALNT2, B2M, NLRC5 or any combination thereof. For example, thegenetically modified cells used to make a tolerizing vaccine orpreparatory regimen can have disrupted GGTA1 only, or disrupted CMAHonly, or disrupted B4GALNT2, B2M or NLRC5 only. The genetically modifiedcells used to make a tolerizing vaccine or preparatory regimen can alsohave disrupted GGTA1 and CMAH, disrupted GGTA1 and B4GALNT2, ordisrupted CMAH and B4GALNT2, or disrupted NLRC5 and B2M. The geneticallymodified cells used to make a tolerizing vaccine or preparatory regimencan have disrupted GGTA1, CMAH, and B4GALNT2. Without wishing to bebound by any particular theory, such disruptions can minimize oreliminate cell-mediated immunity, antibody-mediated immunity,antibody-dependent cell-mediated immunity, and/or cell-dependentantibody-mediated immunity to organ, tissue, cell, and cell line grafts(e.g., xenografts or allografts comprising the same geneticmodification(s) as the cells used in the tolerizing vaccine).

Genetically modified cells used in a tolerizing vaccine or preparatoryregimen can comprise, or further comprise, one or more geneticmodifications that increase expression of one or more genes or geneproducts. The increased expression can be from zero expression, e.g.,the increased expression can be of a gene or gene product that is notnormally expressed in the cell without genetic modification. Theincreased expression can be compared to a threshold level, e.g., a levelnormally expressed in the cell without genetic modification. The geneticmodification(s) can comprise one or more exogenous polynucleotidesencoding a polypeptide (e.g., an endogenous or exogenous polypeptide).

Non-limiting examples of exogenous polynucleotides include, but are notlimited to, polynucleotides encoding one or more of an MHC I formationsuppressor (e.g., an infected cell protein 47 (ICP47)); a regulator ofcomplement activation (e.g., CD46, CD55, or CD59); an inhibitory ligandfor NK cells; a B7 family member (e.g., a programmed death ligand suchas PD-L1 or PD-L2); a serine protease inhibitor (e.g., Spi9); agalectin; an interleukin (e.g., IL-37); a CD40:CD40L blocking agent(e.g., a CD40 antagonist polypeptide, an anti-CD40 ligand polypeptide);a Fas ligand (FasL); any functional fragment thereof; or any combinationthereof. In some embodiments, an inhibitory ligand for NK cells is ahuman leukocyte antigen (HLA), such as human leukocyte antigen E(HLA-E), human leukocyte antigen G (HLA-G), β-2-microglobulin (B2M) orany combination thereof. In some embodiments, the HLA-G is HLA-G1,HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, or any combinationthereof. In some cases, galectins is galectin-1, galectin-2, galectin-3,galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9,galectin-10, galectin-11, galectin-12, galectin-13, galectin-14, orgalectin-15. For example, a galectin can be galectin-9.

Ex Vivo Expansion of Leukocytes

Donor leukocytes can be retrieved from a living donor's spleen. In oneembodiment, B lymphocytes can be taken from the donor in one or moreblood draws and/or apheresis procedures and optionally expanded ex vivo,or a separate cell donor can be identified that is a suitable match orpartial match to the transplant donor. In some cases, separate donorscan be used for the tolerogenic leukocytes and the allograft. Forexample, splenocytes from a cadaveric donor who is fully matched orpartially matched (e.g., haploidentical) with the prospective livingtransplant donor are a clinically viable source of tolerogenicleukocytes. Upon availability of a matched, partially matched, orhaploidentical spleen, a tolerization protocol can be initiated with theinfusion of splenocytes on e.g., day −7 followed by the living donortransplant (e.g., a kidney transplant) on e.g., day 0 and the infusionof ex vivo expanded splenic B cells on e.g., on day +1.

Cells (e.g., leukocytes) used in preparing a tolerizing vaccine/regimenor preparatory regimen can be expanded ex vivo. In some cases, leukocytecells can be expanded in vitro in the presence of one or more reagentsfor a predetermined amount of time prior to use as a tolerizing vaccine.For instance, the cells can be contacted with at least one cytokine forabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 160, 165, 170,175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 390, or 300hours. In some cases, the cells can be contacted with at least onecytokine for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, or more days. In some cases, the cellscan be contacted with at least one cytokine for about 1 to 2, 2 to 3, 3to 4, 1 to 4, 1 to 3, or 2 to 4 weeks. In some cases, the cytokine isone or more interleukin. In some cases, the interleukin (IL) is at leastone of IL-2, IL-4, IL-21, BAFF, multimer CD40L, IL-10, IL-12, and IL-15.In some cases, the cells are contacted with IL-2, IL-4, IL-21, BAFF, andmultimer CD40L.

Leukocytes used in a tolerizing vaccine/regimen or preparatory regimencan comprise at least or at least about 10%, e.g., 25%, CD19 positive,CD20 positive, or CD21 positive MHC Class II positive B cells. Forexample, donor splenocytes can comprise at least or at least about 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%CD19, CD20, and/or CD21 positive MHC Class II positive B cells, e.g.,about, at least, or at least about 10 to 20; 20 to 30; 30 to 40; 40 to50; 50 to 60%, or 60 to 70%. In some cases, splenic B cells orleukocytes used in a tolerizing vaccine/regimen or preparatory regimencan comprise at least or at least about 60%, e.g., 90%, CD19, CD20, orCD21 positive MHC Class II positive B cells. For example, splenic Bcells or leukocytes used in a tolerizing vaccine/regimen or preparatoryregimen can comprise can comprise about, at least, or at least about60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD19, CD20, or CD21 positiveMEW Class II positive B cells e.g., at least or at least about 60 to 70;70 to 80; 80 to 90; or 90 to 95%. In some cases, donor splenocytes orleukocytes used in a tolerizing vaccine/regimen or preparatory regimencan comprise from or from about 50% to 100%, e.g., from or from about60% to 100% or 80% to 100%, CD19, CD20, or CD21 positive MEW Class IIpositive B cells. In some embodiments the MEW class II is swineleukocyte antigen (SLA) Class II. In some embodiments the MEW class IIis human leukocyte antigen (HLA) Class II.

Generating Cells for a Tolerizing Vaccine or Preparatory Regimen

A tolerizing agent of the methods herein can comprise apoptotic cells,non-apoptotic cells, or a combination thereof. Cells for a tolerizingvaccine or preparatory regimen can be made apoptotic a number ofdifferent ways. For example, the cells can be contacted with a chemical(e.g., a fixative or cross-linking agent, a cellular damaging agent, ora combination thereof), to make some or all of the cells apoptotic. Inanother example, the cells can be irradiated (e.g., with ultravioletradiation, gamma radiation, etc.) to make some or all of the cellsapoptotic.

Cells for a tolerizing vaccine or preparatory regimen can be contactedwith a chemical, such as a fixative or cross-linking agent, acarbodiimide, or a carbodiimide derivative, a diimidoester, asulfhydryl-to-sulfhydryl crosslinker, an amine-to-sulfhydrylcrosslinker, a sulfhydryl-to-carbohydrate crosslinker, a photoreactivecrosslinker, an in vivo crosslinker, a cellular damaging agent or anapoptosis inducer. The contacting can make some or all of the cellsapoptotic. Suitable fixatives or cross-linking agents include, but arenot limited to: an amine-to-amine crosslinker, asulfhydryl-to-sulfhydryl crosslinker, an amine-to-sulfhydrylcrosslinker, an in vivo crosslinker, a sulfhydryl-to-carbohydratecrosslinker, a photoreactive crosslinker, a chemoselective ligationcrosslinking agent, a carboxyl-to-amine crosslinker, a carbodiimide,genipin, acrylic aldehyde, diformyl, osmium tetroxide, a diimidoester,mercuric chloride, zinc sulphate, zinc chloride, trinitrophenol (picricacid), potassium dichromate, ethanol, methanol, acetone, acetic acid, ora combination thereof.

The carbodiimide can comprise ethylcarbodiimide; ethylene carbodiimide;N,N′-diisopropylcarbodiimide (DIC); N,N′-dicyclohexylcarbodiimide (DCC);1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, ECDI, orEDAC); or a combination thereof. In some cases, the carbodiimidecomprises ethylcarbodiimide. In some cases, the carbodiimide comprisesethylene carbodiimide. In some cases, the carbodiimide comprisesN,N′-diisopropylcarbodiimide (DIC). In some cases, the carbodiimidecomprises N,N-dicyclohexylcarbodiimide (DCC). In some cases, thecarbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDCI, EDC, ECDI, or EDAC). In some cases, the tolerizing vaccinecomprises cells treated with EDCI derivatives and/or functionalizedEDCI. Methods of preparation, administration and use of tolerizing agente.g., a tolerizing vaccine is well known in the art, for example, seeU.S. Pat. No. 8,734,786B2, U.S. Pat. No. 9,888,673B2.

Cells for a tolerizing vaccine or preparatory regimen can be contactedwith a diimidoester. The contacting can be for a pre-determined time.The contacting can make some or all of the cells apoptotic. Thediimidoester can comprise cyanuric chloride; diisocyanate;diethylpyrocarbonate (DEPC) or diethyl dicarbonate; a maleimide;benzoquinone; or a combination thereof.

Cells for a tolerizing vaccine or preparatory regimen can be contactedwith an amine-to-amine crosslinker. The contacting can be for apre-determined time. The contacting can make some or all of the cellsapoptotic. In some cases, the amine-to-amine-crosslinker comprisesdisuccinimidyl glutarate (DSG); disuccinimidyl suberate (DSS);bis(sulfosuccinimidyl)suberate (BS3); tris-(succinimidyl)aminotriacetate (TSAT); BS(PEG)5; BS(PEG)9; dithiobis(succinimidylpropionate) (DSP); 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP);disuccinimidyl tartrate (DST);bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone (BSOCOES); ethyleneglycol bis(succinimidyl succinate) (EGS); sulfo-EGS; or any combinationthereof. In some cases, the amine-to-amine crosslinker comprises animidoester, such as dimethyl adipimidate (DMA); dimethyl pimelimidate(DMP); dimethyl suberimidate (DMS); dimethyl3,3′-dithiobispropionimidate (DTBP); or any combination thereof. In somecases, the amine-to-amine crosslinker comprises a difluoro, such as1,5-difluoro-2,4-dinitrobenzene (DFDNB).

Cells for a tolerizing vaccine or preparatory regimen can be contactedwith a sulfhydryl-to-sulfhydryl crosslinker. The contacting can be for apre-determined time. The contacting can make some or all of the cellsapoptotic. In some cases, the sulfhydryl-to-sulfhydryl crosslinkercomprises a maleimide, such as bismaleimidoethane (BMOE);1,4-bismaleimidobutane (BMB); bismaleimidohexane (BMH);tris(2-maleimidoethyl)amine (TMEA); BM(PEG)2 (such as1,8-bismaleimido-diethyleneglycol); BM(PEG)3 (such as1,11-bismaleimido-triethyleneglycol), dithiobismaleimidoethane (DTME);or any combination thereof.

Cells for a tolerizing vaccine or preparatory regimen can be contactedwith an amine-to-sulfhydryl crosslinker. The contacting can be for apre-determined time. The contacting can make some or all of the cellsapoptotic. In some cases, the amine-to-sulfhydryl crosslinker comprisesa NHS-haloacetyl crosslinker, a NHS-maleimide, a NHS-pyridyldithiolcrosslinker, a sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) crosslinker, orany combination thereof. The NHS-haloacetyl crosslinkers can comprisesuccinimidyl iodoacetate (SIA); succinimidyl3-(bromoacetamido)propionate (SBAP); succinimidyl(4-iodoacetyl)aminobenzoate (SIAB); sulfo-SIAB; or a combinationthereof. The NHS-maleimide can comprise N-α-maleimidoacet-oxysuccinimideester (AMAS); N-β-maleimidopropyl-oxysuccinimide ester (BMPS);N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS); sulfo-GMBS;m-maleimidobenzoyl-N-hydrosuccinimide ester (MBS); sulfo-MBS; SMCC;sulfo-SMCC; N-ε-malemidocaproyl-oxysuccinimide ester (EMCS); sulfo-EMCS;succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB); sulfo-SMPB;succinimidyl 6-((beta-maleimidopropionamido)hexanoate) (SMPH);sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC);N-κ-maleimidoundecanoyl-oxysulfosuccinimide ester (sulfo-KMUS); or acombination thereof. The NHS-pyridyldithiol crosslinker can comprisesuccinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl6-(3(2-pyridyldithio)propionamido)hexanoate (LC-SPDP), sulfo-LC-SPDP, or4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)tolune (SMPT).

Cells for a tolerizing vaccine or preparatory regimen can be contactedwith a sulfhydryl-to-carbohydrate crosslinker. The contacting can be fora pre-determined time. The contacting can make some or all of the cellsapoptotic. In some cases, the sulfhydryl-to-carbohydrate crosslinkercomprises (N-β-maleimidopropionic acid hydrazide (BMPH),N-ε-maleimidocaproic acid hydrazide (EMCH),4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH),N-κ-maleimidoundecanoic acid hydrazide (KMUH),3-(2-pyridyldithio)propionyl hydrazide (PDPH), or any combinationthereof.

In some cases, the carboxyl-to-amine crosslinker isdicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, EDC, or EDAC),N-hydroxysuccinimide (NHS), sulfo-NHS, or any combination thereof.

Cells for a tolerizing vaccine or preparatory regimen can be contactedwith a photoreactive crosslinker. The contacting can be for apre-determined time. The contacting can make some or all of the cellsapoptotic. In some cases, the photoreactive crosslinker comprises a NHSester/aryl azide, a NHS ester/diazirine, or a combination thereof. TheNHS ester/aryl azide can comprise N-5-azido-2-nitrobenzoyloxysuccinimide(ANB-NOS), sulfo-SANPAH, or a combination thereof. The NHSester/diazirine can comprise SDA (NHS-diazirine/succinimidyl4,4′-azipentanoate), sulfo-SDA, LC-SDA (NHS-LC-diazirine/succinimidyl6-(4,4′-azipentanamido)hexanoate), sulfo-LC-SDA, SDAD(NHS—SS-diazirine/succinimidyl2-((4,4′-azipentanamido)ethyl)1,3′-dithiopropionate), sulfo-SDAD, or acombination thereof.

Cells for a tolerizing vaccine or preparatory regimen can be contactedwith an in vivo crosslinker. The contacting can be for a pre-determinedtime. The contacting can make some or all of the cells apoptotic. The invivo crosslinker can comprise BS3, DTSSP, sulfo-EGS, DSG, DSP, DSS, EGS,sulfo-SDA, sulfo-LC-SDA, sulfo-SDAD, SDA, LC-SDA, SDAD, NHS-esterdiazirine, or any combination thereof.

In some cases, the cells for use in a tolerizing vaccine or preparatoryregimen are treated with a cellular damaging agent or an apoptosisinducer. In some cases, the cellular damaging agent induces apoptosis insome or all of the contacted cells. Non-limiting exemplary cellulardamaging agents include doxorubicin, staurosporine, etoposide,comptothecin, paclitaxel, vinblastine, or any combination thereof.Non-limiting exemplary apoptosis inducers include marinopyrrole A,maritoclax, (E)-3,4,5,4′-tetramethoxystilbene,17-(Allylamino)-17-demethoxygeldanamycin,2,4,3′,5′-tetramethoxystilbene, 2OHOA, 6,8-bis(benzylthio)-octanoicacid, AT101, apoptolidin, FU 40A, ara-G hydrate, arylquin 1, BAD, BAM7,BAX activator molecule 7, BH3I-1, BID, BMS-906024, BV02, bendamustine,borrelidin, borrelidine, cyclopentanecarboxylic acid, NSC 216128,treponemycin, brassinin, brassinine, brefeldin A, ascotoxin, BFA,cyanein, decumbin, bufalin, CCF642, CCT007093, CD437, CHM-1 hydrate,2-(2-fluorophenyl)-6,7-methylenedioxy-2-4-quinolone hydrate, NSC 656158,CIL-102, CP-31398, dihydrochloride hydrate, camalexin,3-(Thiazol-2-yl)-1H-indole, camalexine, carboxyatractyloside,cepharanthine, cepharanthine, cinnabarinic acid, cirsiliol,combretastatin A4, costunolide, DBeQ, DIM-C-pPhtBu, DMXAA, DPBQ,enniatin A1, enniatin A, enniatin B1, enniatin B, erastin, eupatorin,FADD, fluticasone propionate, fosbretabulin disodium, GO-201trifluoroacetate, gambogic acid, HA 14-1, HMBA, hexaminolevulinate(HAL), IMB5046, IMS2186, ikarugamycin, imiquimod, iniparib, kurarinone,LLP-3, lipocalin-2, lometrexol, MI-4F, ML 210, ML291, mollugin,muristerone A, NA-17, NID-1, NPC26, NSC59984, Nap-FF, neocarzinostatin,nifetepimine, nitidine chloride, nutlin-3, nutlin-3a, PKF118-310,PRIMA-1, PRT4165, pemetrexed, penta-O-galloyl-β-D-glucose hydrate,phenoxodiol, prodigiosin (PG), psoralidin, pterostilbene, raltitrexed,raptinal, ridaifen-B, rifabutin, roslin 2, s-p-bromobenzylglutathionecyclopentyl diester, SJ-17255, SMBA1, STF-62247, suprafenacine,syrosingopine, talniflumate, taurolidine, temoporfin, temozolomide,tetrazanbigen, thaxtomin A, thiocolchicine, tirapazamine, UCD38B,UMI-77, undecylprodigiosin, VK3-OCH3, vacquinol-1, violacein, vosaroxin,zerumbone, gAcrp30, gAcrp30/adipolean, or any combination thereof. Cellscontacted with a cellular damaging agent or an apoptosis inducer maysubsequently be contacted with a fixative or cross-linking agent.

Cells for a tolerizing vaccine or preparatory regimen can be madeapoptotic by contacting the cells with a chemical (e.g., a fixative orcross-linking agent, a cellular damaging agent, or a combinationthereof) for a predetermined amount of time. In some embodiments, thecells in the tolerizing vaccine or the preparatory regimen are madeapoptotic by fixing for a predetermined amount time with thecrosslinking agent (e.g., ECDI). In some cases, the predetermined amountof time is about 30 minutes, about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours,or about 72 hours. In some cases, the predetermined amount of time isless than an hour. In some cases, the predetermined time is at leastabout 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40minutes, 50 minutes, 60 minutes, 75, minutes, 90 minutes, 120 minutes,150 minutes, 180 minutes, 210 minutes or 240 minutes. In some cases, thepredetermined time is at most about 30 minutes, 40 minutes, 50 minutes,60 minutes, 75, minutes, 90 minutes, 120 minutes, 150 minutes, 180minutes, 210 minutes or 240 minutes. In some cases, the predeterminedamount of time is about 1 minute to about 240 minutes, 1 minute to about10 minutes, 10 minutes to about 240 minutes, about 10 minutes to about180 minutes, about 10 minutes to about 120 minutes, about 10 minutes toabout 90 minutes, about 10 minutes to about 60 minutes, about 10 minutesto about 30 minutes, about 30 minutes to about 240 minutes, about 30minutes to about 180 minutes, about 30 minutes to about 120 minutes,about 30 minutes to about 90 minutes, about 30 minutes to about 60minutes, about 50 minutes to about 240 minutes, about 50 minutes toabout 180 minutes, about 50 minutes to about 120 minutes, about 50minutes to about 90 minutes, about 50 minutes to about 60 minutes, about10 minutes to about 20 minutes, about 20 minutes to about 30 minutes,about 30 minutes to about 40 minutes, about 40 minutes to about 50minutes, about 50 minutes to about 60 minutes, about 60 minutes to about70 minutes, about 70 minutes to about 80 minutes, about 80 minutes toabout 90 minutes, about 90 minutes to about 100 minutes, about 100minutes to about 110 minutes, about 110 minutes to about 120 minutes,about 10 minutes to about 30 minutes, about 30 minutes to about 50minutes, about 50 minutes to about 70 minutes, about 70 minutes to about90 minutes, about 90 minutes to about 110 minutes, about 110 minutes toabout 130 minutes, about 130 minutes to about 150 minutes, about 150minutes to about 170 minutes, about 170 minutes to about 190 minutes,about 190 minutes to about 210 minutes, about 210 minutes to about 240minutes, up to about 30 minutes, about 30 minutes to about 60 minutes,about 60 minutes to about 90 minutes, about 90 minutes to about 120minutes, or about 120 minutes to about 150 minutes.

The contacting can be at any temperature. In some cases the contactingis performed on ice (e.g., at 4° C.). In other cases, the contacting isperformed at room temperature. In some cases, the contacting isperformed at a temperature of at least about 0° C., 2° C., 4° C., 8° C.,15° C., 20° C., 25, 30° C., 35° C., or 37° C. In some cases, thecontacting is performed at a temperature of at most about 4° C., 8° C.,15° C., 20° C., 25, 30° C., 35° C., 37° C., or 40° C. In some cases, thecontacting is performed at a temperature of about 0° C. to about 37° C.,about 0° C. to about 25° C., about 0° C. to about 15° C., about 0° C. toabout 10° C., about 0° C. to about 8° C., about 0° C. to about 6° C.,about 0° C. to about 4° C., about 0° C. to about 2° C., about 2° C. toabout 10° C., about 2° C. to about 8° C., about 2° C. to about 6° C.,about 4° C. to about 25° C., about 4° C. to about 10° C., about 15° C.to about 37° C., about 15° C. to about 25° C., about 20° C. to about 40°C., about 20° C. to about 37° C., or about 20° C. to about 30° C.

Cells in a tolerizing vaccine or preparatory regimen can aggregate as aresult of the method of making some or all of the cells apoptotic. Forexample, cells can aggregate after contacting with a chemical, such as afixative or crosslinking agent. The predetermined amount of time thatthe cells are contacted with the chemical can be selected to minimizethe amount of aggregation in the tolerizing vaccine or preparatoryregimen. In some cases, aggregates can be removed, for example, bywashing and/or filtration.

In some cases, a tolerizing vaccine or preparatory regimen can comprisefrom or from about 0.01 to 10 aggregates, per μl. For example, thetolerizing vaccine or preparatory regimen can comprise from or fromabout 0.01 to 1, 0.1 to 1, 0.25 to 1, 0.5 to 1, 1 to 5; or 1 to 10aggregate per μl. The tolerizing vaccine or preparatory regimen cancomprise less than about 0.1, 0.5, 0.75, 1, 5, or 10 aggregates per μL.

In some cases, the tolerizing vaccine or preparatory regimen cancomprise less than 5 aggregates per μL. For example, the tolerizingvaccine or preparatory regimen can comprise less than about: 5, 4, 3, 2,1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 aggregates per μL.

In some case, the tolerizing vaccine or preparatory regimen comprises 1or fewer aggregates per μL. For example, the tolerizing vaccine orpreparatory regimen can comprise about 0.01, about 0.05, about 0.1,about 0.2, about 0.3, about 0.4, about 0.5 about 0.6, about 0.7, about0.8, about 0.9, or about 1.0 aggregates per μL.

The tolerizing vaccine or preparatory regimen can include from or fromabout 0.01% to 10%, e.g., from or from about 0.01% to 2%, necroticcells. For example, cells of a tolerizing vaccine or preparatory regimencan comprise from or from about 0.01% to 10%; 0.01% to 7.5%, 0.01% to5%; 0.01% to 2.5%; or 0.01% to 1% necrotic cells. In some embodiments,the cells of a tolerizing vaccine or preparatory regimen of thedisclosure can comprise at most about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, or 10% necrotic cells.

Molecules Conjugated on the Surface of Apoptotic LeukocytesImmunomodulatory Molecules

As used herein, the term “conjugate” or “conjugated to,” and the likerefer to molecular entities (.e.g, peptides of the present disclosureand an apoptotic leucocyte) being linked together through covalent ornon-covalent bonds. Conjugation may be accomplished by directly couplingthe two molecular entities, e.g., creating an ester or amide from anhydroxyl group, amino group, and a carboxylic acid. Conjugation may beaccomplished by indirectly coupling the two molecular entities, e.g.,instituting a linking group such as a polyethylene glycol. Conjugationmay be accomplished by modifying the molecular entities with chemicalgroups that react with one another, e.g., alkyne-functionalized entitywith an azide-functionalized entity or the reduction of thiol groups onindividual entities to form a disulfide bond. Conjugates such asethylene carbodiimide (ECDI), hexamethylene diisocyanate,propyleneglycol di-glycidylether which contain 2 epoxy residues, andepichlorohydrin can be used for fixation of peptides or proteins to theapoptotic leucocyte surface. Reactive carboxyl groups on the surface ofan apoptotic leukocyte can be joined to free amines (e.g., from Lysresidues) on the peptide or protein, by reacting them with, for example,1-ethyl-3-[3,9-dimethyl aminopropyl] carbodiimide hydrochloride (EDC) orN-hydroxysuccinimide ester (NHS). Similarly, the same chemistry may beused to conjugate free amines on the surface of an apoptotic leukocytewith free carboxyls (e.g., from the C-terminus, or from Asp or GIuresidues) on the peptide or protein. Alternatively, free amine groups onthe surface of an apoptotic leuckocyte may be covalently bound topeptides and proteins, or peptide or protein fusion proteins, usingsulfo-SIAB chemistry, essentially as described by Arano et al. (1991)Chem. 2:71-6. A great variety of means, well known in the art, may beused to conjugate the peptides to surface of apoptotic leuckocytes.These methods include any standard chemistry which do not destroy orseverely limit the biological activity of the peptides and that of theapoptotic leuckocytes, and which allow for a sufficient number ofpeptides to be conjugated to the surface in an orientation which allowsfor inducing tolerance. In some embodiments the C-terminal regions of apeptide are conjugated. In other embodiments, the N-terminus of apeptide can be conjugated onto the surface of the apoptotic leucocyte.

In one aspect, the present disclosure provides preparatory regimenand/or tolerizing vaccines and regimen comprising apoptotic cells suchas leucocytes or mesenchymal stromal cells comprising one or morepeptides conjugated to the surface of the apoptotic cell. In some cases,the one or more peptides can comprise an amino acid sequence set forthin any one of SEQ ID NOs. 1-6 and 8-17. In some cases, the peptideconjugated to the surface of an apoptotic cell such as a leukocyte canvary in length. In some cases, the one or more peptides can comprise asequence set forth in any one of SEQ ID NOs. 1-6 and 8-17 and can be atleast about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 275, 200, or 250 amino acids in length. In some cases, the oneor more peptides can comprise a sequence from 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or100% identical to the sequences set forth in any of SEQ ID NOs. 1-6 and8-17. For example, in some cases, the one or more peptides can comprisea sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17 and can beabout 9, about 10, about 1, about 20, about 25, about 30, about 35,about 40, about 45, about 50, about 55, about 60, about 75, about 80,about 90, about 100, about 120, about 140, about 150, about 160, about170, about 180, about 200, about 220, about 250, about 270, about 300,about 330, about 350, about 400, about 440, about 500, about 600, about700, about 800, about 900 or about 1000 amino acids in length. In somecases, the one or more peptides can comprise a sequence set forth in anyone of SEQ ID NOs. 1-6 and 8-17 and be at most about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50,60, 70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length.In some cases, the peptides be about 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 275, 200, or 250 amino acids in length. In someembodiments, the one or more peptides can comprise a sequence set forthin any one of SEQ ID NOs. 1-6 and 8-17 and can be about 5-250, 10-250,20-250, 30-250, 50-250, 100-250, 5-100, 10-100, 20-100, 30-100, 50-100,5-50, 10-50, 20-50, 30-50, 5-30, 10-30, 15-30, or 20-30 amino acids inlength.

In some embodiments, the peptides conjugated on the surface of apoptoticleukocytes are derived from a MHC molecule. The term “MHC molecule”refers to a molecule comprising Major Histocompatibility Complex (MHC)glycoprotein protein sequences. The term “MHC” as used herein will beunderstood to refer to the Major Histocompability Complex, which isdefined as a set of gene loci specifying major histocompatibilitycomplex glycoprotein antigens including the human leukocyte antigen(HLA). The term “HLA” as used herein will be understood to refer toHuman Leukocyte Antigens, which is defined as the majorhistocompatibility antigens found in humans. As used herein, “HLA” isthe human form of “MHC” and therefore can be used interchangeably.Examples of HLA proteins that can be utilized in accordance with thepresently disclosed and claimed inventive concept(s) include, but arenot limited to, an HLA class I α chain, an HLA class II α chain and anHLA class II β chain. Specific examples of HLA class II α and/or βproteins that may be utilized in accordance with the presently disclosedand claimed inventive concept(s) include, but are not limited to, thoseencoded at the following gene loci: HLA-DRA; HLA-DRB1; HLA-DRB3,4,5;HLA-DQA; HLA-DQB; HLA-DPA; and HLA-DPB.

In some embodiments, the apoptotic cells further comprise one or morepeptides derived from a MHC class I molecule. As such, the sequences ofamino acid residues in the peptide can be substantially similar orfunctionally comparable to a polypeptide sequence in the MHC molecule.Thus, “a peptide derived from a MHC class II molecule” refers to apeptide that has a sequence “from a region in an MHC class II molecule”(e.g., the hypervariable region), and is a peptide that has a sequence70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%,98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to the naturallyoccurring MHC amino acid sequence of the region. In some embodiments,the peptide derived from a MHC class II molecule can comprise a sequencefrom 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%,98%, 98.5%, 99%, 99.5%, 99.9%, 99.95% or 100% identical to thehypervariable region of the WIC class II molecule. Thus, “a peptidederived from a MHC class I molecule” refers to a peptide that has asequence “from a region in an WIC class I molecule” (e.g., thehypervariable region), and is a peptide that has a sequence 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%,99%, 99.5%, 99.9%, 99.95% or 100% identical to the naturally occurringWIC amino acid sequence of the region. In some embodiments, the peptidederived from a MHC class I molecule will comprise a sequence from thehypervariable region of the MHC class I molecule.

The peptides derived from a recipient's MHC class II molecule maycomprise an entire WIC class II molecule. The peptides derived from arecipient's WIC class II molecule may comprise an entire α chain of DR,DQ or DP. The peptides derived from a recipient's MHC class II moleculemay comprise entire β chain of DR, DQ, or DP. The peptides derived froma recipient's WIC class II molecule may comprise entire α1 and/or α2domains of DR, DQ or DP. The peptides derived from a recipient's WICclass II molecule may comprise entire β1 and/or β2 domains of DR, DQ, orDP. The peptides derived from a recipient's WIC class II molecule maycomprise MHC-DR1, MHC-DR2, MHC-DR3, MHC-DR4, and/or MHC-DRS.

The peptides derived from a recipient's MHC class II molecule maycomprise a fragment of an α1 and/or α2 domain of DR, DQ or DP. Thepeptides derived from a recipient's WIC class II molecule may comprise afragment of a β1 and/or β2 domain of DR, DQ, or DP. The peptides derivedfrom a recipient's WIC class II molecule may comprise a fragment ofMHC-DR1, MHC-DR2, MHC-DR3, MHC-DR4, and/or MHC-DRS. The peptides derivedfrom a recipient's MHC class II molecule may comprise a sequence from ahypervariable region. The peptides derived from a recipient's WIC classII molecule can comprise an in silico-identified high, medium, or lowaffinity peptides from the hypervariable region of the DRB molecule(e.g., a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidpeptide).

The peptides derived from a recipient's MHC class II molecule may besynthesized or recombinant. In some cases, the peptides derived from arecipient's MHC class II molecule may between about 10 and 30 aminoacids in length. The peptides derived from a recipient's MHC class IImolecule may be at least 10 to 30 amino acids in length. In someembodiments, the peptides derived from a recipient's MHC class IImolecule can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60,70, 80, 90, 100, 125, 150, 275, 200, or 250 amino acids in length. Insome embodiments, the peptides derived from a recipient's MHC class IImolecule can be at most about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 275, 200, or 250 amino acids in length. In some embodiments,the peptides derived from a recipient's MHC class II molecule can beabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150,275, 200, or 250 amino acids in length. In some embodiments, thepeptides derived from a recipient's MHC class II molecule can be about5-250, 10-250, 20-250, 30-250, 50-250, 100-250, 5-100, 10-100, 20-100,30-100, 50-100, 5-50, 10-50, 20-50, 30-50, 5-30, 10-30, 15-30, or 20-30amino acids in length.

Cells (e.g., leukocytes) for a tolerizing vaccine or preparatory regimencan be treated with a fixative or crosslinking agent (e.g., acarbodiimide such as ECDI) in the presence of one or more antigensand/or epitopes. In some cases, the epitopes can comprise a peptidecontaining an amino acid sequence set forth in any one of SEQ ID NOs.1-6 and 8-17. In some cases, the antigens and/or epitopes can compriseantigens and/or epitopes from a transplant donor, a transplantrecipient, a third party, or a combination thereof. In some cases, thecells in the tolerizing vaccine or preparatory regimen are coupled torecipient antigens and/or epitopes. In some cases, the cells in thetolerizing vaccine or preparatory regimen are coupled to third partyantigens and/or epitopes. In some cases, the cells in the tolerizingvaccine or preparatory regimen are coupled to transplant donor antigensand/or epitopes.

In some embodiments, a “cocktail” of peptides can be conjugated on thesurface of the apoptotic leucocytes. In some embodiments, the apoptoticcells such as leucocytes can comprise one or more peptides derived fromMHC class II molecule. In some embodiments, the apoptotic cells such asleucocytes can comprise, for example, at least 2, 3, 5, 7, 10, 15, 20,30, 40, 50, 100 or more peptides derived from a MHC class II molecule.In some embodiments, the apoptotic leucocytes can comprise one or morepeptides derived from a MHC class I molecule. In some embodiments, theapoptotic cells such as leucocytes can comprise, for example, at least2, 3, 5, 7, 10, 15, 20, 30, 40, 50, 100 or more peptides derived from aMHC class I molecule. A mixture of more than one peptide derived from aMHC class II molecule, has the advantage of inducing increased immunetolerance response in the recipient. The increased tolerance can be, forexample, through a mechanism called linked supersession. The mechanismof linked suppression will be known to an artisan skilled in the art.For instance, peptides comprising sequences from hypervariable regionsof α and β chains may be used in combination.

In certain embodiments, the size of a protein or a peptide that isconjugated to the surface of the apoptotic leukocyte may comprise, butis not limited to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750,775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300,1400, 1500, 1750, 2000, 2250, 2500 amino molecules or greater, includingany range or value derivable therein, or derivative thereof. It iscontemplated that peptides might be altered by fusing or conjugating aheterologous peptide sequence with a particular function (e.g., forpresentation as a protein complex, for enhanced immunogenicity, etc.).

Cells (e.g., leukocytes) for a tolerizing vaccine or preparatory regimencan be treated with a fixative or crosslinking agent (e.g., acarbodiimide such as ECDI) in the presence of one or moreimmunomodulatory molecules. In some cases, the one or moreimmunomodulatory molecules can comprise one or more peptides comprisingan amino acid sequence set forth in any one of SEQ ID NOs. 1-6 and 8-17.In some cases, the one or more immunomodulatory molecules can compriseall or a portion of: IFN-γ, an NF-kB inhibitor, vitamin D3, siCD40,anti-CD40 antibody, cobalt protoporphyrin, insulin B9-23,α1-antitrypsin, a cluster of differentiation protein, a gp39 antagonist,α1-antitrypsin, CD47, PD-L1, PD-L2, CTLA-4, rapamycin, compstatin,abatacept, ipilimumab, or a combination thereof. The NF-kB inhibitor cancomprise dehydroxymethylepoxyquinomicin (DHMEQ), curcumin, triptolide,Bay-117085, or a combination thereof. The cluster of differentiationprotein can comprise CD4, CD46, CD47, CD55, CD59, or a portion thereof,or a combination thereof.

In some embodiments, the one or more immunomodulatory molecules cancomprise all or a portion of a calcineurin inhibitor (e.g., cyclosporineor tacrolimus), a costimulatory signal blockade, an IL-2 signalinginhibitor (e.g., daclizumab or basiliximab), a cell cycle blocker (e.g.,mycophenolate mofetil (MMF) or azathioprine), a T cell recirculationinhibitor (e.g., FTY720 or another sphingosine 1-phosphate (S1P)receptor agonist), a nitrogen mustard alkylating agent (e.g.,cyclophosphamide), a complement C3 or C5 inhibitor, or any combinationthereof.

In some embodiments, the immunomodulatory molecules can target T cellreceptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxic Tlymphocyte associated protein 4 (CTLA-4), programmed cell death protein1 (PD-1, e.g., pembrolizumab), programmed death ligand 1 (PD-L1, e.g.,MPDL3280A), CD40L (CD154), CD40, inducible costimulatory (ICOS), IL-2,TNF-α (e.g., infliximab), IL-6 or IL-6R (e.g., tocilizumab, actemra,clazakizumab, ALD518, siltuximab, elsilimomab, sirukumab, sarilumab,olokizumab), IL-7, CD2, CD20, CD52, α-4 integrin, mTOR (e.g., rapamycinor everolimus), DNA synthesis, molecules in pro-inflammatory pathways(e.g., cytokines, α1-antitrypsin, NFkB), or any combination thereof.

Cells (e.g., leukocytes) for tolerizing vaccine or preparatory regimencan be treated with a fixative or crosslinking agent (e.g., acarbodiimide such as ECDI) in the presence of an agent that increasesexpression of anti-inflammatory cytokines in a recipient. In some cases,the agent can comprise one or more peptides comprising an amino acidsequence set forth in any one of SEQ ID NOs. 1-6 and 8-17. In somecases, the anti-inflammatory cytokines may include, for example, TGF-β,IL-10, IL-13, or a combination thereof. In some cases, the agent thatincreases expression of anti-inflammatory cytokines in the recipientcomprises α1-antitrypsin.

In some cases where ADLs are administered to a transplant recipientmultiple times, ADLs from all the doses can all be conjugated with thesame immunomodulatory molecules, agents that increases expression ofanti-inflammatory cytokines, and/or antigens or epitopes. In some caseswhere ADLs are administered to a transplant recipient multiple times,ADLs from one or more doses can be conjugated with a first set ofimmunomodulatory molecules, agents that increases expression ofanti-inflammatory cytokines, and/or antigens or epitopes, and ADLs fromother dose(s) can be conjugated with a different set of immunomodulatorymolecules, agents that increases expression of anti-inflammatorycytokines, and/or antigens or epitopes.

Short Term Immunosuppression

In some cases, a preparatory regimen or tolerizing vaccine of thedisclosure can comprise administering one or more immunosuppressionagents and/or anti-inflammatory agents to a transplant recipient. Forexample, in some cases, the regimen may include administering one ormore immunosuppression agents and/or anti-inflammatory agents inaddition to apoptotic cells (such as apoptotic donor leukocytes ormesenchymal stromal cells). In other cases, the regimen may includeadministering one or more immunosuppression agents and/oranti-inflammatory agents in addition to an anti-CD40 agent, for e.g., apeptide comprising an amino acid sequence with at least 80%, at least85%, at least 90% or at least 95% sequence identity to the sequences setforth in any one of SEQ ID NOs. 1-6 and 8-17.

In some embodiments, the short term immunosuppression comprisesadministering an antagonistic anti-CD40 agent. An anti-CD40 agent or ananti-CD40 ligand agent can be, for example, a peptide comprising anamino acid sequence with at least 80%, at least 85%, at least 90% or atleast 95% sequence identity to the sequences set forth in any one of SEQID NOs. 1-6 and 8-17. In some cases, the anti-CD40 agent or anti-CD40ligand agent can be, for example, a peptide comprising an amino with atleast 80%, 85%, 88%, 90%, 93%95% or 99% sequence identity to thesequences set forth in any one of SEQ ID NOs. 1-6 and 8-17.

In some cases, an anti-CD40 agent or an anti-CD40 ligand agent can be,for example, an antagonistic anti-CD40 antibody, and antagonisticanti-CD40 ligand antibody, or an antigen binding fragmented thereof. Ananti-CD40 agent or an anti-CD40 ligand agent can be an antagonisticanti-CD40 antibody or antigen-binding fragment thereof. Non-limitingexamples of antagonistic anti-CD40 antibodies include 2C10, 2C10R4,ASKP1240, 4D11, bleselumab, BI-655064, HCD122, CFZ533, ch5D12, CDP7657,and FFP104. In some cases, the anti-CD40 agent is ASKP1240. An anti-CD40agent or an anti-CD40 ligand agent can be an antagonistic anti-CD40ligand antibody or antigen-binding fragment thereof. Non-limitingexamples of antagonistic anti-CD40 ligand antibodies include BG9588,ruplizumab, toralizumab, IDEC-131, dapirolizumab, letolizumab,BMS-986004, VIB4920, and MEDI4920.

In some cases, an immunosuppression agent used for short termimmunosuppression can be any one or more of MMF (mycophenolate mofetil(Cellcept)), ATG (anti-thymocyte globulin), anti-CD154 (CD40L),alemtuzumab (Campath), CTLA4-Ig (Abatacept/Orencia), belatacept(LEA29Y), daclizumab (Ze-napax), basiliximab (Simulect), infliximab(Remicade), cyclosporin, deoxyspergualin, soluble complement receptor 1,cobra venom factor, compstatin, anti C5 antibody (eculizumab/Soliris),methylprednisolone, FTY720, everolimus, anti-CD154-Ab, leflunomide,anti-IL-2R-Ab, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40antibody, and anti-CD122 antibody, human anti-CD154 monoclonal antibody,CD40 antagonist, and CD40L (CD154) antagonist.

In some cases, an immunosuppression agent used for short termimmunosuppression can target T cell receptor (TCR), CD3e, FK506-bindingprotein 12 (FKBP12), cytotoxic T lymphocyte associated protein 4(CTLA-4), programmed cell death protein 1 (PD-1), programmed deathligand 1 (PD-L1), CD40L (CD154), CD40, inducible costimulatory (ICOS),IL-2, TNF-α, IL-6, IL-7, CD2, CD20, CD52, α-4 integrin, mTOR(mechanistic target of rapamycin, everolimus, serolimus), DNA synthesis,or any combination thereof. In some cases, an immunosuppression agentused for short term immunosuppression can be a MHC/TCR interactionblockade, a nonselective depleting agent, calcineurin inhibitor,costimulatory signal blockade, cytokine blockade, lymphocyte depletingagent, cell adhesion inhibitor, IL-2 signaling inhibitor, cell cycleblocker, or any combination thereof. For example, the MHC/TCRinteraction blockade can be anti-abTCR mAb T10B9. For example, thenonselective depleting agent can be anti-CD3 mAb (OKT3) or antithymocyteglobulin (ATG). For example, the calcineurin inhibitor can becyclosporine or tacrolimus. For example, the costimulatory signalblockade can be anti-CTLA-4 mAb, abatacept, ipilimumab, anti-PD-1 (suchas pembrolizumab), anti-PD-L1 (such as MPDL3280A), anti-CD154 mAb,anti-CD40 mAb, or anti-ICOS mAb. For example, the cytokine blockade canbe anti-CD25 mAb (such as daclizumab or basiliximab), anti-TNF(etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab),anti-IL-6/IL-6R mAb (such as clazakizumab, ALD518, siltuximab,elsilimomab, sirukumab, olokizumab, sarilumab, tocilizumab, actemra), oranti-IL-7 mAb. For example, the lymphocyte depleting agent can beanti-CD2 mAb, fusion protein with IgG1 (such as alefacept), anti-CD20mAb (such as rituximab), or anti-CD52 mAb (such as alemtuzumab). Forexample, the cell adhesion inhibitor can be anti-very large antigen 4(VLA4) (such as natalizumab). For example, the IL-2 signaling inhibitorcan be sirolimus (rapamycin) or everolimus. For example, the cell cycleblocker can be mycophenolate mofetil (MMF) or azathioprine.

Short term immunosuppression can comprise administering one or moreimmunosuppression agents and/or anti-inflammatory agents of thedisclosure for at most about 100 days after a transplant. Short termimmunosuppression can comprise administering one or moreimmunosuppression agents and/or anti-inflammatory agents of thedisclosure, for example, for at most about 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32,33, 34, 35, 40, 42, 49, 50, 55, 60, 70, 80, 90, or 100 days after atransplant. In some cases, short term immunosuppression can concludewithin 28 days after a transplant. In some cases, short termimmunosuppression can conclude about 21 days after a transplant.

In some embodiments, short term immunosuppression can begin prior totransplantation. For example, short term immunosuppression can commenceabout or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 25, 28, 29, 30, 35, or50 days prior to a transplant. In some cases, short termimmunosuppression can commence at most about 10 days prior to atransplant. In some cases, short term immunosuppression can commenceabout 7 days prior to a transplant.

The duration of short term immunosuppression (e.g., the length of timebetween administering a first dose and a final dose of animmunosuppression agents and/or anti-inflammatory agents can be about orat most about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 25, 28, 29, 30, 31, 32, 33, 34, 35, 40, 42, 49, 50,55, 60, 70, 80, 90, or 100 days. In some cases, the duration of shortterm immunosuppression can be about or at most about 30 days. In somecases, the duration of short term immunosuppression can be about 28days.

Administration of Compositions in Combination with a Tolerizing Agent

In one aspect, a peptide or compositions disclosed herein can beadministered as a tolerizing agent. In another embodiment, a peptide orcompositions disclosed herein can be administered before, after, and/orduring the administration of a tolerizing agent. In some cases, thepeptide of compositions disclosed herein can be administered between day−100 and day 0, e.g., on day −90, day −80, day −70, day −60, day −50,day −40, day −30, day −20, day −15, day −14, day −13, day −12, day −11,day −10, day −9, day −8, day −7, day −6, day −5, day −4, day −3, day −2,day −1, or day 0 relative to the administration of a tolerizing agent.In some cases, the peptide or compositions disclosed herein can beadministered concomitantly with the tolerizing agent. In some cases, thepeptide or compositions disclosed herein can be administered on or onabout day −100 to −50; −50 to −40; −40 to −30; −30 to −20; −20 to −10;−10 to −5; −7 to −1, relative to the administration of a tolerizingagent.

In some cases, the peptide or composition disclosed herein can beadministered between day 0 and day 100, e.g., on day 100, day 90, day80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4,day 3, day 2 or day 1 relative to the administration of a tolerizingagent. For example, the peptide or compositions disclosed herein can beadministered on or on about day 100 to 50; 50 to 40; 40 to 30; 30 to 20;20 to 10; 10 to 5; 7 to 1, relative to the administration of atolerizing agent.

In some cases, the peptide or compositions disclosed herein can beadministered between day 0 and day 300, e.g., on day 300, day 200, day100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6,day 5, day 4, day 3, day 2 or day 1 relative to the administration of atolerizing agent. For example, the peptide or compositions disclosedherein can be administered on or on about day 300 to 200; 200 to 100;100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1,relative to the administration of a tolerizing agent.

In some cases, a peptide or compositions disclosed herein can beadministered between day 0 and day 600, e.g., on day 600, day 500, day400, day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50,day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10,day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1 relativeto the administration of a tolerizing agent. For example, a peptide orcomposition disclosed herein can be administered on or on about day 600to 500; day 500 to 400; day 400 to 300; 300 to 200; 200 to 100; 100 to50; 50 to 40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative tothe administration of a tolerizing agent.

In some cases, a peptide or compositions disclosed herein can beadministered between day 0 and day 1,000, e.g., on day 1,000, day 900,day 800, day 700, day 600, day 500, day 400, day 300, day 200, day 100,day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15,day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day5, day 4, day 3, day 2 or day 1 relative to the administration of atolerizing agent. For example, the a peptide or compositions disclosedherein can be administered on or on about day 1,000 to 900; 900 to 800;800 to 700; 700 to 600; 600 to 500; day 500 to 400; day 400 to 300; 300to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to 10;10 to 5; 7 to 1, relative to the administration of a tolerizing agent.

In some cases, a peptide or compositions disclosed herein can beadministered on the day when a tolerizing agent is administered. Inother cases, a peptide or can be administered before and after theadministration of the tolerizing vaccine or preparatory regimen.

A peptide or compositions disclosed herein can be administered before,after, and/or during the day of the transplantation of the graft cell,tissue, or organ. In some cases, the peptide of compositions disclosedherein can be administered between day −100 and day 0, e.g., on day −90,day −80, day −70, day −60, day −50, day −40, day −30, day −20, day −15,day −14, day −13, day −12, day −11, day −10, day −9, day −8, day −7, day−6, day −5, day −4, day −3, day −2, day −1, or day 0 relative to the dayof the transplantation of the graft cell, tissue, or organ. In somecases, the peptide or compositions disclosed herein can be administeredconcomitantly with the day of the transplantation of the graft cell,tissue, or organ. In some cases, the peptide or compositions disclosedherein can be administered on or on about day −100 to −50; −50 to −40;−40 to −30; −30 to −20; −20 to −10; −10 to −5; −7 to −1, relative to theday of the transplantation of the graft cell, tissue, or organ.

In some cases, the peptide or composition disclosed herein can beadministered between day 0 and day 100, e.g., on day 100, day 90, day80, day 70, day 60, day 50, day 40, day 30, day 20, day 15, day 14, day13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4,day 3, day 2 or day 1 relative to the day of the transplantation of thegraft cell, tissue, or organ. For example, the peptide or compositionsdisclosed herein can be administered on or on about day 100 to 50; 50 to40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the dayof the transplantation of the graft cell, tissue, or organ.

In some cases, the peptide or compositions disclosed herein can beadministered between day 0 and day 300, e.g., on day 300, day 200, day100, day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day15, day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6,day 5, day 4, day 3, day 2 or day 1 relative to the day of thetransplantation of the graft cell, tissue, or organ. For example, thepeptide or compositions disclosed herein can be administered on or onabout day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to20; 20 to 10; 10 to 5; 7 to 1, relative to the day of thetransplantation of the graft cell, tissue, or organ.

In some cases, a peptide or compositions disclosed herein can beadministered between day 0 and day 600, e.g., on day 600, day 500, day400, day 300, day 200, day 100, day 90, day 80, day 70, day 60, day 50,day 40, day 30, day 20, day 15, day 14, day 13, day 12, day 11, day 10,day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1 relativeto the day of the transplantation of the graft cell, tissue, or organ.For example, a peptide or composition disclosed herein can beadministered on or on about day 600 to 500; day 500 to 400; day 400 to300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20to 10; 10 to 5; 7 to 1, relative to the day of the transplantation ofthe graft cell, tissue, or organ.

In some cases, a peptide or compositions disclosed herein can beadministered between day 0 and day 1,000, e.g., on day 1,000, day 900,day 800, day 700, day 600, day 500, day 400, day 300, day 200, day 100,day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 20, day 15,day 14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day5, day 4, day 3, day 2 or day 1 relative to the day of thetransplantation of the graft cell, tissue, or organ. For example, the apeptide or compositions disclosed herein can be administered on or onabout day 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500;day 500 to 400; day 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to40; 40 to 30; 30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to the dayof the transplantation of the graft cell, tissue, or organ.

In some cases, a peptide or compositions disclosed herein can beadministered on the day of the transplantation of the graft cell,tissue, or organ. In other cases, a peptide or can be administeredbefore and after the day of the transplantation of the graft cell,tissue, or organ.

A preparatory regimen can comprise multiple doses of a peptide orcomposition described herein, before, and/or during and/or aftertransplantation of a graft cell, tissue, or organ. In some cases, apreparatory regimen can comprise multiple doses of a peptide comprisinga sequence with at least 80%, 85% 90% or 95% sequence identity set forthin any one of SEQ ID NOs. 1-6 and 8-17, before, and/or during and/orafter transplantation of a graft cell, tissue, or organ. A tolerizingregimen can comprise multiple doses of a peptide or compositiondescribed herein, before, and/or during and/or after transplantation ofa graft cell, tissue, or organ. In some cases, a tolerizing regimen cancomprise multiple doses of a peptide comprising a sequence with at least80%, 85% 90% or 95% sequence identity set forth in any one of SEQ IDNOs. 1-6 and 8-17, before, and/or during and/or after transplantation ofa graft cell, tissue, or organ. The multiple doses can be referred to ascomprising an initial dose and one or more booster doses. The initialdose can occur prior to or concurrently with the transplant of the graftcell tissue or organ. The booster dose(s), when administered, occurafter the initial dose. Depending upon when the initial dose of thetolerizing vaccine is administered, one or more booster doses can beadministered before, and/or concurrently with, and/or after transplantof the graft cell, tissue, or organ.

Subsequent (e.g., booster) dose(s) of a preparatory regimen ortolerizing vaccine can be administered in any interval of time followinga preceding dose (e.g., an initial dose). For example, the subsequentdose can be administered about 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 90 days, 120days, 150 days, or 180 days after the preceding dose.

Depending upon when the initial dose is administered subsequent(booster) dose(s) can be administered before, concurrently with, orafter transplantation of the graft cell, tissue, or organ. In somecases, the preparatory regimen comprises at least one dose, at least twodoses, at least three doses of a preparatory regimen or tolerizingvaccine prior to transplantation.

In some cases, two doses of the tolerizing vaccine or preparatoryregimen can be administered. The first dose can be administered, forexample, on day −14, −10, −9, −8, −7, −6, −5, −4, −3, −2, −1, 0, 1, 2,3, 4, 5, 6, or 7 relative to transplant of donor cells, organs, and/ortissues on day 0. The second dose can be administered, for example, onday −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 relative totransplant of donor cells, organs, and/or tissues on day 0. In someembodiments, the first dose is administered on day −8 and the seconddose is administered on day −1. In some embodiments, the first dose isadministered on day −8 and the second dose is administered on day 0. Insome embodiments, the first dose is administered on day −8 and thesecond dose is administered on day 1. In some embodiments, the firstdose is administered on day −7 and the second dose is administered onday −1. In some embodiments, the first dose is administered on day −7and the second dose is administered on day 0. In some embodiments, thefirst dose is administered on day −7 and the second dose is administeredon day 1. In some embodiments, the first dose is administered on day −12and the second dose is administered on day −4. In some embodiments, thefirst dose is administered on day −11, −12, −13, or −14 and the seconddose is administered on day −3, −4, −5, or −6.

In some cases, a second dose of the tolerizing vaccine/preparatoryregimen (e.g., a booster vaccine) can be administered on day 100, day90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28, day27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19, day18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10, day9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1, relative totransplant of donor cells, organs, and/or tissues on day 0. For example,the second dose of the tolerizing vaccine (e.g., a booster vaccine) canbe administered 1 day after transplant of donor cells, organs, and/ortissues. In some cases, a second dose of a tolerizing vaccine is givenconcomitantly on day 0 with transplant donor cells, organs, and/ortissues. In some cases, a second dose of a tolerizing vaccine is notrequired.

In some cases, three doses of the tolerizing vaccine/preparatory regimenor preparatory regimen can be administered. The first dose can beadministered, for example, on day −14, −10, −9, −8, −7, −6, −5, −4, −3,−2, −1, 0, 1, 2, 3, 4, 5, 6, or 7 relative to transplant of donor cells,organs, and/or tissues on day 0. The second dose can be administered,for example, on day −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28relative to transplant of donor cells, organs, and/or tissues on day 0.The third dose can be administered, for example, on day 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180,200, 250, 300, 350, or 365 relative to transplant of donor cells,organs, and/or tissues on day 0. In some embodiments, the first dose isadministered on day −8, the second dose is administered on day 1, andthe third dose is administered on day 7. In some embodiments, the firstdose is administered on day −8, the second dose is administered on day1, and the third dose is administered on day 14. In some embodiments,the first dose is administered on day −8, the second dose isadministered on day 1, and the third dose is administered on day 21. Insome embodiments, the first dose is administered on day −7, the seconddose is administered on day 1, and the third dose is administered on day7. In some embodiments, the first dose is administered on day −7, thesecond dose is administered on day 1, and the third dose is administeredon day 14. In some embodiments, the first dose is administered on day−7, the second dose is administered on day 1, and the third dose isadministered on day 21.

In some cases, a third dose of the tolerizing vaccine/preparatoryregimen (e.g., a booster vaccine) can be administered on day 300, day200, day 100, day 90, day 80, day 70, day 60, day 50, day 40, day 30,day 29, day 28, day 27, day 26, day 25, day 24, day 23, day 22, day 21,day 20, day 19, day 18, day 17, day 16, day 15, day 14, day 13, day 12,day 11, day 10, day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2or day 1, relative to transplant of donor cells, organs, and/or tissueson day 0. For example, the tolerizing vaccine can be administered on oron about day 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30to 20; 20 to 10; 10 to 5; or 7 to 1, relative to transplant of donorcells, organs, and/or tissues on day 0.

In some cases, a fourth dose of the tolerizing vaccine/preparatoryregimen (e.g., a booster vaccine) can be administered on day 600, day500, day 400, day 300, day 200, 100, day 90, day 80, day 70, day 60, day50, day 40, day 30, day 29, day 28, day 27, day 26, day 25, day 24, day23, day 22, day 21, day 20, day 19, day 18, day 17, day 16, day 15, day14, day 13, day 12, day 11, day 10, day 9, day 8, day 7, day 6, day 5,day 4, day 3, day 2 or day 1, relative to transplant of donor cells,organs, and/or tissues on day 0. For example, the tolerizing vaccine canbe administered on or on about day 600 to 500; 500 to 400; 400 to 300;300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30; 30 to 20; 20 to10; 10 to 5; 7 to 1, relative to transplant of donor cells, organs,and/or tissues on day 0.

In some cases, a fifth dose of the tolerizing vaccine or preparatoryregimen (e.g., a booster vaccine) can be administered on day 1,000, day900, day 800, day 700, day 600, day 500, day 400, day 300, day 200, 100,day 90, day 80, day 70, day 60, day 50, day 40, day 30, day 29, day 28,day 27, day 26, day 25, day 24, day 23, day 22, day 21, day 20, day 19,day 18, day 17, day 16, day 15, day 14, day 13, day 12, day 11, day 10,day 9, day 8, day 7, day 6, day 5, day 4, day 3, day 2 or day 1,relative to transplant of donor cells, organs, and/or tissues on day 0.For example, the tolerizing vaccine can be administered on or on aboutday 1,000 to 900; 900 to 800; 800 to 700; 700 to 600; 600 to 500; 500 to400; 400 to 300; 300 to 200; 200 to 100; 100 to 50; 50 to 40; 40 to 30;30 to 20; 20 to 10; 10 to 5; 7 to 1, relative to transplant of donorcells, organs, and/or tissues on day 0.

Administration of the tolerizing vaccine or preparatory regimen canresult in long term tolerance to the cell, tissue, or organ transplantin the transplant recipient. In some cases, the long term tolerance isfor a period of at least one month, at least two months, at least threemonths, at least four months, at least five months, at least six months,at least seven months, at least eight months, at least nine months, atleast ten months, at least eleven months, at least twelve months, atleast thirteen months, at least fourteen months, at least fifteenmonths, at least sixteen months, at least seventeen months, at leasteighteen months, at least nineteen months, at least twenty months, atleast twenty-one months, at least twenty-two months, at leasttwenty-three months, or at least twenty-four months. In some cases, thelong term tolerance is for a period of at least 1 year, at least 2years, at least 3 years, at least 4 years, at least 5 years, at least 6years, at least 7 years, at least 8 years, at least 9 years, or at least10 years. In some cases, the long term tolerance is achieved in theabsence of a booster vaccine or booster regimen. In some cases, the longterm tolerance is achieved with an administration of a booster vaccineor booster regimen in one or multiple doses. In some cases, one or morebooster vaccine doses are administered on the day of, or at least 1 day,2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days,27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days,35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days,43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days,51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days,59 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days,95 days, 100 days, 105 days, 110 days, 115 days, 120 days, 125 days, 130days, 135 days, 140 days, 145 days, 150 days, 155 days, 160 days, 165days, 170 days 175 days, 180 days, 185 days, 190 days, 195 days, 200days, 205 days, 210 days, 215 days, 220 days, 230 days or 240 days afterthe transplantation. In certain specific cases, one or more (forinstance three) doses of a preparatory regimen is administered prior totransplantation, and one or more booster vaccine doses are provided 1,7, 14, 21, 90, or up to 180 days after transplantation.

A tolerizing vaccine or preparatory regimen can be administered with orwithout an adjuvant (e.g., one or more immunomodulatory molecules). Insome cases, the adjuvant enhances the tolerogenic properties of thetolerizing vaccine by inhibiting activation and maturation of antigenpresenting cells.

A dose of a tolerizing vaccine or preparatory regimen can vary basedupon the weight of a recipient of a tolerizing vaccine. For example, thedose of the tolerizing vaccine or preparatory regimen can compriseabout: 1×101 cells/kg, 1×102 cells/kg, 1×103 cells/kg, 1×104 cells/kg,1×105 cells/kg, 1×106 cells/kg, 1×107 cells/kg, 1×108 cells/kg, 1×109cells/kg, 1×1010 cells/kg, 1×1011 cells/kg, 1×1012 cells/kg, or more. Insome cases, a dose of the tolerizing vaccine or preparatory regimen cancomprises about: 1×101 to 1×102 cells/kg; 1×102 to 1×103 cells/kg; 1×103to 1×104 cells/kg; 1×104 to 1×105 cells/kg; 1×105 to 1×106 cells/kg;1×106 to 1×107 cells/kg; 1×107 to 1×108 cells/kg; 1×108 to 1×109cells/kg; 1×109 to 1×1010 cells/kg, 1×1010 to 1×1011 cells/kg, 1×1011 to1×1012 cells/kg. For example, a dose of the tolerizing vaccine orpreparatory regimen for administration can be about 0.01×109 cells/kg,0.02×109 cells/kg, 0.03×109 cells/kg, 0.04×109 cells/kg, 0.05×109cells/kg, 0.06×109 cells/kg, 0.07×109 cells/kg, 0.08×109 cells/kg,0.09×109 cells/kg, 0.1×109 cells/kg, 0.2×109 cells/kg, 0.21×109cells/kg, 0.22×109 cells/kg, 0.23×109 cells/kg, 0.24×109 cells/kg,0.25×109 cells/kg, 0.26×109 cells/kg, 0.27×109 cells/kg, 0.28×109cells/kg, 0.29×109 cells/kg, 0.3×109 cells/kg, 0.4×109 cells/kg, 0.5×109cells/kg, 0.6×109 cells/kg, 0.7×109 cells/kg, 0.8×109 cells/kg, 0.9×109cells/kg, 1.0×109 cells/kg, 1.5×109 cells/kg, 2.0×109 cells/kg, 2.5×109cells/kg, 3.0×109 cells/kg, 3.5×109 cells/kg, 4.0×109 cells/kg, 4.5×109cells/kg, 5.0×109 cells/kg, 5.5×109 cells/kg, 6.0×109 cells/kg, 6.5×109cells/kg, 7.0×109 cells/kg, 7.5×109 cells/kg, 8.0×109 cells/kg, 8.5×109cells/kg, 9.0×109 cells/kg, 9.5×109 cells/kg, 10.0×109 cells/kg, or25.0×109 cells/kg.

In some cases, a dose of the tolerizing vaccine or preparatory regimencan comprise at least about: 1×104 cells/kg, 5×104 cells/kg, 1×105cells/kg, 5×105 cells/kg, 1×106 cells/kg, 5×106 cells/kg, 1×107cells/kg, 5×107 cells/kg, 1×108 cells/kg, 2×108 cells/kg, 3×108cells/kg, 4×108 cells/kg, 5×108 cells/kg, 6×108 cells/kg, 7×108cells/kg, 8×108 cells/kg, 9×108 cells/kg, 1×109 cells/kg, 1×1010cells/kg, or more.

The methods herein can comprise administering at least or at least about0.25×109 cells (e.g., apoptotic donor leukocytes (ADLs), such asECDI-treated cells, e.g., ECDI-treated leukocytes, or apoptoticmesenchymal stromal cells) per kg recipient body weight. For example, atleast or at least about 1×107, 1×108, 0.25×109, 0.50×109, 0.75×109,1.00×109, 1.25×109, 1.50×109, 1.75×109 or 2×109 cells (e.g.,ECDI-treated cells, e.g., ECDI-treated leukocytes) per kg recipient bodyweight ECDI-treated cells can be administered.

The cells can comprise leukocytes, e.g., splenocytes, peripheral bloodmononuclear cells (PBMCs), stem-cell derived leukocytes, or acombination thereof. The splenocytes, PBMCs, stem-cell derivedleukocytes, or the combination thereof can comprise B cells or Blymphocytes. The cells can comprise primary cells, cells expanded exvivo, cells of a cell line, or a combination thereof. The cells cancomprise mesenchymal stromal cells.

Cells of tolerizing vaccine/regimen or preparatory regimen for each doseof administration can be suspended in a volume suitable for transfusion.For example, the cells can be suspended in a volume of about: 0.1 ml,0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23ml, 24 ml, 25 ml, 26 ml, 27 ml, 28 ml, 29 ml, 30 ml, 31 ml, 32 ml, 33ml, 34 ml, 35 ml, 36 ml, 37 ml, 38 ml, 39 ml, 40 ml, 41 ml, 42 ml, 43ml, 44 ml, 45 ml, 46 ml, 47 ml, 48 ml, 49 ml, 50 ml, 60 ml, 70 ml, 80ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, or 500 ml. For example, thecells of tolerizing vaccine or preparatory regimen for each dose ofadministration can be suspended in a volume of about: 0.1 ml to 1 ml; 1ml to 10 ml; 10 ml to 50 ml; 50 ml to 100 ml; 100 ml to 200 ml; 200 mlto 300 ml; 300 ml to 400 ml; or 400 ml to 500 ml. For example, 75×106cells of tolerizing vaccine or preparatory regimen can be suspended in avolume of 0.5 ml.

Tolerizing vaccines/regimen or preparatory regimens can be administered(e.g., by intravenous infusion) in a volume that varies depending uponthe weight of the recipient. For example, the tolerizing vaccine orpreparatory regimen can be given intravenously in a volume of at leastor at least about 0.01 ml, 0.1 ml, 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml,10 ml, 20 ml, 30 ml, 40 ml or 50 ml per kg recipient body weight, e.g.,at least or at least about 0.01 to 0.1, 0.1 to 1, 1 to 2; 2 to 3; 3 to4; 4 to 5; 1 to 5; 5 to 10; 10 to 20; 20 to 30; 30 to 40; or 40 to 50 mlper kg recipient body weight. In some cases, the tolerizing vaccine(e.g., comprising ECDI-treated cells) is given intravenously in a volumeof about 7 ml per kg recipient body weight.

Booster doses of a tolerizing vaccine or preparatory regimen cancomprise fewer cells than an initial dose of the tolerizing vaccine orpreparatory regimen. For example, a booster or subsequent dose of thetolerizing vaccine or preparatory regimen can comprise about: 1%, 2%,3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% fewercells, or less than the initial or preceding dose of the tolerizingvaccine or preparatory regimen.

A cell of a tolerizing vaccine or a preparatory regimen can have acirculation half-life after it is administered to a subject. In somecases, a tolerizing vaccine or preparatory regimen described herein canhave a circulation half-life of at least or at least about 0.1, 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, 24, 36, 48, 60, or 72 hours. Forexample, the circulation half-life of the tolerizing vaccine orpreparatory regimen cells can be from or from about 0.1 to 0.5; 0.5 to1.0; 1.0 to 2.0; 1.0 to 3.0; 1.0 to 4.0; 1.0 to 5.0; 5 to 10; 10 to 15;15 to 24; 24 to 36; 36 to 48; 48 to 60; or 60 to 72 hours. In somecases, a tolerizing vaccine or preparatory regimen described herein canhave a circulation half-life of at least or at least about 3 hours.

The cells of the tolerizing vaccine or preparatory regimen can betreated to enhance their circulation half-life. Such treatment caninclude coating the cell with a protein, e.g., CD47. The cell treated toenhance its circulation half-life can be a non-apoptotic cell. The celltreated to enhance its circulation half-life can be an apoptotic cell.Alternatively, the cell in a tolerizing vaccine or preparatory regimencan be genetically modified (e.g., insertion of a transgene such as CD47in its genome) to enhance its circulation half-life. The cellgenetically modified to enhance its circulation half-life can be anon-apoptotic cell. The cell genetically modified to enhance itscirculation half-life can be an apoptotic cell.

The tolerizing vaccine or the preparatory regimen can be advantageous intransplantation, for example, in xenotransplantation or inallotransplantation, by tolerizing a graft recipient and preventing ordelaying graft rejection. The tolerization or the preparatory regimencan be conferred to a graft recipient without the use ofimmunosuppressive therapies (e.g., one or more immunomodulatorymolecules). However, in some cases, other immunosuppressive therapiescan be used in combination with tolerizing vaccines to prevent,decrease, or delay transplantation rejection.

The dose of a peptide or composition thereof required in presence of atolerizing agent can be lower than the dose of a peptide or compositiondisclosed herein required in its absence by at least or at least about5%. For example, dose of a peptide or composition disclosed herein canbe lower by at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, ormore. The term “lower” and its grammatical equivalents as used hereincan refer to using less peptide or compositions thereof compared to arequired dose when one or more wild-type cells, organs, and/or tissuesis transplanted into a recipient (e.g., a human or a non-human animal).

Transplant Survival

In some embodiments, the methods disclosed herein can increase theduration of survival of a transplant (e.g., a xenograft or an allografttransplant) in a recipient for a period of at least one month, at leasttwo months, at least three months, at least four months, at least fivemonths, at least six months, at least 1 year, at least 2 years, at least3 years, at least 4 years, at least 5 years, at least 6 years, at least7 years, at least 8 years, at least 9 years, or at least 10 years.

In some cases, the transplant survives for a period of at least onemonth, at least two months, at least three months, at least four months,at least five months, at least six months, at least seven months, atleast eight months, at least nine months, at least ten months, at leasteleven months, at least twelve months, at least thirteen months, atleast fourteen months, at least fifteen months, at least sixteen months,at least seventeen months, at least eighteen months, at least nineteenmonths, at least twenty months, at least twenty-one months, at leasttwenty-two months, at least twenty-three months, or at least twenty-fourmonths. In some cases, the transplant survives for a period of at least1 year, at least 2 years, at least 3 years, at least 4 years, at least 5years, at least 6 years, at least 7 years, at least 8 years, at least 9years, or at least 10 years. In some cases, the survival is achieved inthe absence of a booster vaccine or booster regimen. In some cases, thetransplant survival is achieved with an administration of a booster dosein one or multiple doses.

Different doses of a peptide or compositions disclosed herein can begiven to a recipient before, after, and/or during transplant of donorcells, organs, and/or tissues to induce donor-specific tolerance in arecipient. In some cases, a first dose of a peptide or compositionsdisclosed herein can comprise at least or at least about 1 mg, 5 mg, 10mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or200 mg of the a peptide or compositions disclosed herein per kgrecipient body weight. In certain cases, a first dose of a peptide orcompositions disclosed herein can comprise at least or at least about 30mg, 40 mg, 50 mg, 60 mg, 70 mg of a peptide or compositions disclosedherein per kg recipient body weight. In some cases, a first dose of apeptide or compositions disclosed herein can comprise from or from about1 mg to 200 mg, e.g., from or from about 20 mg to 100 mg; 30 mg to 80mg; 30 mg to 70 mg; 40 mg to 70 mg; 40 mg to 60 mg; 50 mg to 70 mg; or60 mg to 80 mg of the a peptide or compositions disclosed herein per kgrecipient body weight.

Immunosuppressive Agents

Methods of the present disclosure further comprises administering animmunomodulatory agent to the recipient. The immunomodulatory agent canbe for example, an immunosuppressive agent. The term “immunosuppressiveagent” as used herein refers to substances that act to suppress or maskthe immune system of the host into which the graft is beingtransplanted. This would include substances that suppress cytokineproduction, down-regulate or suppress self-antigen expression, or maskthe MHC antigens.

Thus, peptides or compositions of the disclosure can be used alone or insome embodiments in combination with immunosuppressive agent thatinhibits rejection of a transplant. The immunosuppressive agent can beadministered simultaneously or at a separate time than the peptide orcompositions herein. Non-limiting examples of immunosuppressive agentsare azathioprine, corticosteroids, cyclosporine,2-amino-6-aryl-5-substituted pyrimidines; azathioprine orcyclophosphamide; bromocryptine; glutaraldehyde; antiidiotypicantibodies for MHC antigens; cyclosporin A; one or more steroids,preferably corticosteroids and glucocorticosteroids such as prednisone,methyl prednisolone, and dexamethasone; anti-interferon-gammaantibodies; anti-tumor necrosis factor-alpha antibodies; anti-tumornecrosis factor-beta antibodies; anti-interleukin-2 antibodies;anticytokine receptor antibodies such as anti-IL-2 receptor antibodies;heterologous anti-lymphocyte globulin; pan-T antibodies, preferablyOKT-3 monoclonal antibodies; antibodies to CD4; streptokinase;streptodomase; or RNA or DNA from the host.

In some cases, the immunosuppressive agent inhibits T cell activation.The immunosuppressive agent that inhibits T cell activation can be ananti-CD40 agent or anti-CD40L (CD154) agent. In some embodiment, theanti-CD40 agent can be an anti-CD40 antibody. The anti-CD40 antibody canbe an antagonistic antibody. The anti-CD40 antibody can be a Fab′anti-CD40L monoclonal antibody fragment CDP7657. The anti-CD40 antibodycan be a FcR-engineered, Fc silent anti-CD40L monoclonal domainantibody.

In some cases, the peptide or compositions disclosed herein isadministered with one or more additional immunosuppression agentdescribed herein, such as further comprising providing to the recipientone or more of an anti-CD40 agent or anti-CD40L (CD154) agent (e.g., ananti-CD40 antibody), a B-cell targeting agent (e.g., B cell depletingbiologics, for example, a biologic targeting CD20, CD19, or CD22, and/orB cell modulating biologics, for example, a biologic targeting BAFF,BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), an mTOR inhibitor, aTNF-alpha inhibitor, a IL-6 inhibitor, a nitrogen mustard alkylatingagent (e.g., cyclophosphamide), a complement C3 or C5 inhibitor, IFNγ,an NFκB inhibitor, α1-antitrypsin, vitamin D3, siCD40, cobaltprotoporphyrin, insulin B9-23, a cluster of differentiation protein(e.g., CD46, CD55, or CD59), any combination thereof, or any fragmentthereof. In some cases, the NFκB inhibitor isdehydroxymethylepoxyquinomicin (DHMEQ), curcumin, triptolide,Bay-117085, or a combination thereof. In some cases, B-cell targetingbiologic can be Rituximab, anti-CD20 antibody.

In some cases, immunosuppressive agent can be MMF (mycophenolate mofetil(Cellcept)), ATG (anti-thymocyte globulin), anti-CD154 (CD40L),alemtuzumab (Campath), B-cell targeting agent (e.g., B cell depletingbiologics, for example, a biologic targeting CD20, CD19, or CD22, and/orB cell modulating biologics, for example, a biologic targeting BAFF,BAFF/APRIL, CD40, IgG4, ICOS, IL-21, B7RP1), anti-IL-6R antibody(tocilizumab, Actemra), anti-IL-6 antibody (sarilumab, olokizumab),CTLA4-Ig (Abatacept/Orencia), belatacept (LEA29Y), mTOR inhibitor(sirolimus (e.g., Rapamune), rapamycin, everolimus), tacrolimus(Prograf), daclizumab (Zenapax), basiliximab (Simulect), infliximab(Remicade), cyclosporin, deoxyspergualin, soluble complement receptor 1,cobra venom factor, compstatin, anti C5 antibody (eculizumab/Soliris),methylprednisolone, FTY720, everolimus, anti-CD154-Ab, leflunomide,anti-IL-2R-Ab, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40antibody, and anti-CD122 antibody, human anti-CD154 monoclonal antibody,CD40 antagonist, and CD40L (CD154) antagonist. Non-limiting examples ofB-cell targeting biologics include antagonistic anti-CD40 mAb antibody,Fc-engineered anti-CD40L antibodies, Rituximab, anti-CD20 antibody. Oneor more than one immunosuppressive agents/drugs can be used together orsequentially. One or more than one immunosuppressive agents/drugs can beused for induction therapy or for maintenance therapy. The same ordifferent drugs can be used during induction and maintenance stages. Forexample, daclizumab (Zenapax) is used for induction therapy andtacrolimus (Prograf) and sirolimus (Rapamune), or everolimus, or anyother mTOR inhibitor is used for maintenance therapy. In anotherexample, daclizumab (Zenapax) is used for induction therapy and low dosetacrolimus (Prograf) and low dose sirolimus (Rapamune) is used formaintenance therapy. Immunosuppression can also be achieved usingnon-drug regimens including, but not limited to, whole body irradiation,thymic irradiation, and full and/or partial splenectomy. Thesetechniques can also be used in combination with one or moreimmunosuppressive drug.

In some embodiments, one or more immunomodulatory molecules can target Tcell receptor (TCR), CD3e, FK506-binding protein 12 (FKBP12), cytotoxicT lymphocyte associated protein 4 (CTLA-4), programmed cell deathprotein 1 (PD-1), programmed death ligand 1 (PD-L1), CD40L (CD154),CD40, inducible costimulatory (ICOS), IL-2, TNF-α, IL-6, IL-7, CD2,CD20, CD52, α-4 integrin, mTOR (mechanistic target of rapamycin,everolimus, serolimus), DNA synthesis, or any combination thereof. Insome embodiments, the one or more immunomodulatory molecule can target Bcell, (e.g., B cell depleting biologics, for example, a biologictargeting CD20, CD19, or CD22, and/or B cell modulating biologics, forexample, a biologic targeting BAFF, BAFF/APRIL, CD40, IgG4, ICOS, IL-21,B7RP1). In some cases, the B cell targeting agent can be anti-CD20 mAb(such as rituximab) or other B-cell depleting antibody. In someembodiments, the immunosuppressive drugs can be a MHC/TCR interactionblockade, a nonselective depleting agent, calcineurin inhibitor,costimulatory signal blockade, cytokine blockade, lymphocyte depletingagent, cell adhesion inhibitor, IL-2 signaling inhibitor, cell cycleblocker, or any combination thereof. For example, the MHC/TCRinteraction blockade can be anti-abTCR mAb T10B9. For example, thenonselective depleting agent can be anti-CD3 mAb (OKT3) or antithymocyteglobulin (ATG). For example, the calcineurin inhibitor can becyclosporine or tacrolimus. For example, the costimulatory signalblockade can be anti-CELA-4 mAb, abatacept, ipilimumab, anti-PD-1 (suchas pembrolizumab), anti-PD-L1 (such as MPDL3280A), anti-CD154 mAb,anti-CD40 mAb, or anti-ICOS mAb. For example, the cytokine blockade canbe anti-CD25 mAb (such as daclizumab or basiliximab), anti-TNF(infliximab), anti-IL-6 mAb (such as ALD518), or anti-IL-7 mAb. Forexample, the lymphocyte depleting agent can be anti-CD2 mAb, fusionprotein with IgG1 (such as alefacept), anti-CD20 mAb (such asrituximab), or anti-CD52 mAb (such as alemtuzumab). For example, thecell adhesion inhibitor can be anti-very large antigen 4 (VLA4) (such asnatalizumab). For example, the IL-2 signaling inhibitor can be sirolimus(rapamycin) or everolimus. For example, the cell cycle blocker can bemycophenolate mofetil (MMF) or azathioprine.

The doses, time and mode of administration of a peptide or compositionsdisclosed herein or an immunomodulatory agent can be easily determinedby one of skill in the art. The key factor in selecting an appropriatedose and scheduling is the result obtained, i.e., graft survivallong-term. For example, a relatively high dose may be needed eitherinitially for the treatment of hyperacute graft rejection, which can beattributed to antibody-mediated graft destruction, or at a later stagecharacterized by a sudden decline in graft function.

When an immunosuppressive agent is used, it may be administered by anysuitable means, including parenteral, and, if desired for localimmunosuppressive treatment, intralesionally. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, andsubcutaneous administration. In addition, when an immunosuppressiveagent is used, it is suitably administered by pulse infusion,particularly with declining doses, or by continuous infusion.

In one aspect, provided herein is a compositions disclosed herein canfurther comprise an immunomodulatory agent, for example, animmunosuppressive agent. An “effective amount” of an immunosuppressiveagent and peptides disclosed herein is an amount which achieves a degreeof immunosuppression sufficient to delay, inhibit, suppress or moderatetissue transplant rejection and/or delay, inhibit, suppress or moderateone or more symptoms of an inflammatory disease e.g., autoimmune diseasedescribed herein. Preferably, the peptides of the present disclosure incombination with an immunomodulatory agent are used at dosages suitablefor inducing transplant survival or, alternatively, at dosages at whichthe immunosuppressive activities of the two agents synergize.Accordingly, sub-therapeutic dosages can be used in the disclosedmethods, i.e., dosages which are lower than the amounts that would beeffective when the peptide or immunomodulatory agent is used alone.Suitable sub-therapeutic dosages of one or both agents of the disclosedcombination therapy are those which are sufficient, when the two agentsare used in combination, to delay, inhibit, suppress or moderate anundesired immune response to an antigen, or inhibit transplant rejectionas a result as described above. The skilled artisan will be able todetermine such dosages using ordinary experimentation, such as by usinganimal models. One advantage of the disclosed combined therapy,therefore, is that the subject being treated can in some instances bespared the side-effects of higher levels of immunosuppression resultingfrom, for example, corticosteroids and inhibitors of calcineurin.

Additional Therapeutic Agents

Of course, the peptides or compositions of the present disclosure can beused in combination with other therapies for treating autoimmunedisorders, inflammation, cancer or for inhibiting transplant rejection.Accordingly, the compositions and methods of the present disclosure cancomprise an additional therapeutic agent. The therapeutic agent can be,for example, anti-inflammatory agent, immunosuppressive agent,anti-tumor agent as relevant for the condition to be treated.Immunosuppressive agents have been described above.

Non-limiting example of anti-tumor agents include antibiotics andanalogs (e.g., aclacinomycins, actinomycin f1, anthramycin, azaserine,bleomycins, cactinomycin, carubicin, carzinophilin, chromomycins,dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,epirubicin, idarubicin, menogaril, mitomycins, mycophenolic acid,nogalamycin, olivomycines, peplomycin, pirarubicin, plicamycin,porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin,zinostatin, zorubicin), antimetabolites (e.g. folic acid analogs (e.g.,denopterin, edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®,trimetrexate), purine analogs (e.g., cladribine, fludarabine,6-mercaptopurine, thiamiprine, thioguanine), pyrimidine analogs (e.g.,ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil,gemcitabine, tagafur).

Anti-inflammatory agent can be Steroidal Anti-inflammatory Agents, suchas 21-acetoxypregnenolone, alclometasone, algestone, amcinonide,beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol,clobetasone, clocortolone, cloprednol, corticosterone, cortisone,cortivazol, deflazacort, desonide, desoximetasone, dexamethasone,diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort,flucloronide, flumethasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandrenolide, fluticasone propionate, formocortal, halcinonide,halobetasol propionate, halometasone, halopredone acetate,hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone,medrysone, meprednisone, methylprednisolone, mometasone furoate,paramethasone, prednicarbate, prednisolone, prednisolone25-diethylamino-acetate, prednisolone sodium phosphate, prednisone,prednival, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide, triamcinolone benetonide, and triamcinolonehexacetonide and the like. Anti-inflamatory agent can be Non-Steroidalantiinflammatory agents such as Aminoarylcarboxylic acid derivatives(e.g., enfenamic acid, etofenamate, flufenamic acid, isonixin,meclofenamic acid, mefenamic acid, niflumic acid, talniflumate,terofenamate, tolfenamic acid), arylacetic acid derivatives (e.g.,aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil,bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac,felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac,indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid,mofezolac, oxametacine, pirazolac, proglumetacin, sulindac, tiaramide,tolmetin, tropesin, zomepirac), arylbutyric acid derivatives (e.g.,bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (e.g.,clidanac, ketorolac, tinoridine), arylpropionic acid derivatives (e.g.,alminoprofen, benoxaprofen, bermoprofen, bucloxic acid, carprofen,fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam,indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen,pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid,ximoprofen, zaltoprofen), pyrazoles (e.g., difenamizole, epirizole),pyrazolones (e.g., apazone, benzpiperylon, feprazone, mofebutazone,morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propyphenazone,ramifenazone, suxibuzone, thiazolinobutazone), salicylic acidderivatives (erg., acetaminosalol, aspirin, benorylate, bromosaligenin,calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisicacid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate,mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine,parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide,salicylamide o-acetic acid, salicylsulfuric acid, salsalate,sulfasalazine), thiazinecarboxamides (e.g., ampiroxicam, droxicam,isoxicam, lornoxicam, piroxicam, tenoxicam), ε-acetamidocaproic acid,s-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine,bendazac, benzydamine, α-bisabolol, bucolome, difenpiramide, ditazol,emorfazone, fepradinol, guaiazulene, nabumetone, nimesulide, oxaceprol,paranyline, perisoxal, proquazone, superoxide dismutase, tenidap, andzileuton and the like.

In some embodiments, the therapeutic agent can be a biologic. As usedherein a biologic refers to any biological material suitable for atherapeutic or in vivo diagnostic purpose. Biologic therapeutics includepeptides, proteins, vaccines, antibodies, aptamers, nucleic acids, DNA,RNA, antisense oligonucleotides, viruses and bacteria.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with, a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorderassociated with a CD40 activity, such as, but not limited to, ainflammation, autoimmune disease or a cancer. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of at leastslowing of progress or worsening of symptoms that would be expected inabsence of treatment. Beneficial or desired clinical results include,but are not limited to, alleviation of one or more symptom(s),diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. The term “treatment” of adisease also includes providing relief from the symptoms or side-effectsof the disease (including palliative treatment).

The methods of the disclosure, including treating a CD40 associateddisorder of a subject, likely results in an improvement in the subjects'condition, a reduction of symptoms or decreasing the subject's risk fordeveloping symptoms associated with a CD40 associated disorder.Improvements therefore include one or more decreased symptoms associatedwith autoimmunity, allergy, cancer, etc. An improvement may also bereducing the frequency or amount of a drug used for treating a subjecthaving or at risk of having a CD40 associated disorder. For example,autoimmune patients treated with steroids may require less steroid whentreated in combination with a peptide or compositions disclosed herein.An improvement therefore would include reducing the dosage frequency oramount of steroid that the subject was administered in comparison to thedosage frequency or amount administered prior to treatment with apeptide or compositions disclosed herein. An improvement may be, forexample, longer transplant survival, or reduction of side effectsassociated with conventional immunosuppressive regiments fortransplantation.

An improvement may be relatively short in duration, e.g., several hours,days or weeks, or extend over a longer period of time, e.g., months oryears. The improvement need not be a complete ablation of any or allsymptoms of the disorder. For example, reducing severe rheumatoidarthritis to a less severe form is an improvement. Thus, a satisfactoryclinical endpoint is achieved when there is an incremental improvementor a partial reduction in the subject's condition or associatedsymptoms, over a short or long duration.

The term “effective amount” as used herein refers to the amount neededto alleviate at least one or more symptom of the disease or disorder,and relates to a sufficient amount of pharmacological composition toprovide the desired effect (e.g., binding to CD40 and/or inhibition ofthe interaction of CD40 and CD154). The term “therapeutically effectiveamount” therefore refers to an amount that is sufficient to effect aparticular effect when administered to a typical subject. An effectiveamount as used herein would also include an amount sufficient to delaythe development of a symptom of the disease, alter the course of asymptom disease (for example but not limited to, slow the progression ofa symptom of the disease), or reverse a symptom of the disease. Thus, itis not possible to specify the exact “effective amount”. For any givencase, however, an appropriate “effective amount” can be determined byone of ordinary skill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the antibody or antigen binding fragment thereof),which achieves a half-maximal inhibition of symptoms as determined incell culture, or in an appropriate animal model. Levels in plasma can bemeasured, for example, by high performance liquid chromatography. Theeffects of any particular dosage can be monitored by a suitablebioassay. The dosage can be determined by a physician and adjusted, asnecessary, to suit observed effects of the treatment.

The compositions of the disclosure are administered in effectiveamounts. An “effective amount” is that amount of a composition thatalone, or together with further doses, produces the desired response. Inthe case of transplantation, a desired response is inhibition oftransplant rejection or increasing transplant survival. In the case oftreating a particular disease, such as arthritis, the desired responseis inhibiting the progression of the disease. This may involve onlyslowing the progression of the disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently. This can be monitored by routine methods.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the disorder, the activity of the specificcompound, the route of administration, the rate of clearance of thecomposition, the duration of treatment, the drugs used in combination orcoincident with the compositions, the age, body weight, sex, diet, andgeneral health of the subject, and like factors well known in themedical arts and sciences. Various general considerations taken intoaccount in determining the “therapeutically effective amount” are knownto those of skill in the art and are described, e.g., in Gilman et al,eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics,8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Co., Easton, Pa., 1990. These factors are wellknown to those of ordinary skill in the art and can be addressed with nomore than routine experimentation. It is generally preferred that amaximum dose of the individual components or combinations thereof beused, that is, the highest safe dose according to sound medicaljudgment. The compositions used in the foregoing methods preferably aresterile and contain an effective amount of the active agents forproducing the desired response in a unit of weight or volume suitablefor administration to a patient.

Kits

In another embodiment of the disclosure, an article of manufacture whichcontains the pharmaceutical composition in a solution form or in alyophilized form or a kit comprising an article of manufacture isprovided. The kit can comprise instructions for diluting the compositionor for its reconstitution and/or use. The article of manufacturecomprises a container. Suitable containers include, for example,bottles, vials (e.g. dual chamber vials), syringes (such as dual chambersyringes) and test tubes. The container may be formed from a variety ofmaterials such as glass or plastic. The container holds the lyophilizedformulation and a label on, or associated with, the container mayindicate directions for reconstitution and/or use. For example, thelabel may indicate that the lyophilized formulation is reconstituted topeptide concentrations as described above. The label may furtherindicate that the formulation is useful or intended for subcutaneousadministration. The container holding the formulation may be a multi-usevial, which allows for repeat administrations (e.g., from 2-6administrations) of the reconstituted formulation. The article ofmanufacture may further comprise a second container comprising asuitable diluent (e.g., BWFI). Upon mixing of the diluent and thelyophilized formulation, the final protein concentration in thereconstituted formulation will generally be at least 50 mg/mL. Thearticle of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

Therapeutic kits may have a single container which contains theformulation of the peptide pharmaceutical compositions with or withoutother components (e.g., other compounds or pharmaceutical compositionsof these other compounds) or may have distinct container for eachcomponent. In some embodiments, therapeutic kits of the disclosureinclude a formulation of peptide disclosed herein or compositionscomprising said peptides or an acid addition salt thereof as disclosedherein packaged for use in combination with the co-administration of asecond compound (such as an anti-inflammatory agent, immunomodulatingagent, anti-tumor agent, a natural product, a hormone or antagonist, aanti-angiogenesis agent or inhibitor, a apoptosis-inducing agent or achelator) or a pharmaceutical composition thereof. The components of thekit may be pre-complexed or each component may be in a separate distinctcontainer prior to administration to a patient. The components of thekit may be provided in one or more liquid solutions, preferably, anaqueous solution, more preferably, a sterile aqueous solution. Thecomponents of the kit may also be provided as solids, which may beconverted into liquids by addition of suitable solvents, which arepreferably provided in another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain apparatus (e.g., oneor more needles, syringes, eye droppers, pipette, etc.), which enablesadministration of the peptides of the disclosure which are components ofthe present kit.

In another embodiment of the disclosure, an article of manufacture whichcontains compositions comprising apoptotic leukocytes and an anti-CD40agent comprising one or more peptides with an amino sequence with atleast 85%, at least 90% or at least 95% sequence identity to thesequences set forth in any one of SEQ ID NOs. 1-6 and 8-17, in asolution form or in a lyophilized form or a kit comprising an article ofmanufacture is provided. The kits of the instant disclosure can be foruse in transplantation of a transplant in a recipient or reducing orinhibiting occurrence of GVHD in a recipient. In some embodiments, thekit is useful as a preparatory regimen prior to the transplantation. Insome embodiments, the kit is useful as a tolerizing regimenpost-transplantation. The kit can comprise instructions for diluting thecomposition or for its reconstitution and/or use. The article ofmanufacture comprises one or more containers. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Thecontainer holds the compositions e.g., in a lyophilized or solution formand a label on, or associated with, the container may indicatedirections for reconstitution and/or use. For example, the label mayindicate that the lyophilized composition is reconstituted to aneffective amount as described above. The label may further indicate thatthe composition is useful or intended for subcutaneous administration orintravenous administration. The container holding the composition may bea multi-use vial, which allows for repeat administrations (e.g., from2-6 administrations) of the composition. The article of manufacture mayfurther comprise a container comprising a suitable diluent (e.g., BWFI).Upon mixing of the diluent and the composition, the final concentrationin the composition can be, for example, an effective amount suitable foradministration. The article of manufacture may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

Kits may have a single container which contains, for example apoptoticleukocytes fixed in a crosslinking agent with or without othercomponents (e.g., and an anti-CD40 agent or an anti-CD40 ligand agent).In some cases, the anti-CD40 agent conjugated to the apoptoticleukocytes may comprise a peptide comprising an amino acid sequence withat least 85%, at least 90% or at least 95% sequence identity to thesequences set forth in any one of SEQ ID NOs. 1-6 and 8-17. Thecomponents of the kit may be pre-complexed or each component may be in aseparate distinct container prior to administration to a patient. Thecomponents of the kit may be provided in one or more liquid solutions,preferably, an aqueous solution, more preferably, a sterile aqueoussolution. The components of the kit may also be provided as solids,which may be converted into liquids by addition of suitable solvents,which are preferably provided in another distinct container. In someembodiments, kits of the disclosure include the components disclosedherein packaged for use in combination with the co-administration of anadditional component (such as an anti-inflammatory agent,immunomodulating agent, anti-tumor agent, a natural product, a hormoneor antagonist, an anti-angiogenesis agent or inhibitor, aapoptosis-inducing agent, or a chelator). The container of a kit may bea vial, test tube, flask, bottle, syringe, or any other means ofenclosing a solid or liquid. In some embodiments, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a kit will contain apparatus (e.g., one or moreneedles, syringes, eye droppers, pipette, etc.), which enablesadministration of the compositions of the disclosure which arecomponents of the present kit.

In some embodiments, the kit disclosed herein further comprises thetransplant. In some embodiments, the transplant is a kidney, liver,heart, lung, pancreas, islet cell, small bowel, bone marrow,hematopoietic stem cell, embryonic or induced pluripotent stemcell-derived islet beta cell, embryonic or induced pluripotent stemcell-derived islet, embryonic or induced pluripotent stem cell-derivedhepatocyte or a combination thereof. In some embodiments, the transplantcan be autologous, allograft, or a xenograft.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1. Identification of CD40 Binding Peptides that could Block CD40Mediated Costimulation

Table 1 lists the amino acid sequences of CD40 binding peptides. Acontrol CD40-binding peptide, Peptide 1 was included in the studiesdescribed below for comparison. Peptide 1 has the amino acid sequence ofVLQWAEKGYYTMSNN (SEQ ID NO: 7). In silico prediction using the AminoAcid Complement Wave method was used to identify peptides 2-7 thatexhibited high affinity to the human CD40 epitope. Peptides that canbind CD40 and block the CD40-CD40L signaling cascade were identified.Sequences of the peptides are presented in the table below. Theidentified peptides were synthesized with and without fluorescentlabeling to study the binding and functional properties of the peptidesto interfere in the CD40-CD40L signaling. Peptides 8-17 are exemplaryfragments of peptide 2-7 expected to exhibit high affinity to the humanCD40 epitope.

TABLE 1 lists the amino acid sequences of the peptidesidentified to bind CD40. SEQ ID NO: 1 VLQWAERGFYTTSNN Peptide 2SEQ ID NO: 2 LLQWAERGYFMTSNN Peptide 6 SEQ ID NO: 3 VVQWAEKGFFTMSNNPeptide 3 SEQ ID NO: 4 VLNWADKAYFTMSQN Peptide 4 SEQ ID NO: 5LVNWADKAFYTTSNN Peptide 5 SEQ ID NO: 6 VVQWAEKGYYTMSQN Peptide 7SEQ ID NO: 8 VLQWAERGFY Peptide 8 SEQ ID NO: 9 LLQWAERGYF Peptide 9SEQ ID NO: 10 VVQWAEKGFF Peptide 10 SEQ ID NO: 11 VLNWADKAYF Peptide 11SEQ ID NO: 12 LVNWADKAFY Peptide 12 SEQ ID NO: 13 RGFYTTSNN Peptide 13SEQ ID NO: 14 RGYFMTSNN Peptide 14 SEQ ID NO: 15 KGFFTMSNN Peptide 15SEQ ID NO: 16 KAYFTMSQN Peptide 16 SEQ ID NO: 17 KAFYTTSNN Peptide 17

Example 2. Screening of CD40-Binding Peptides for Binding to MononuclearCells

In order to screen the ability of the cyclic peptides to bind to CD40,peripheral blood mononuclear cells (PBMC) purified from 2 Rhesusmacaques (RM) were incubated with 504 of fluorochrome tagged CD40interacting peptides, Peptide 1-7 listed in Table 1 for 30 minutes at 4°C. The cells were washed 3× in PBS and analyzed by flow cytometry.Stained cells were acquired on a 3-laser BD Canto II instrument (BDBioscience). A minimum of 200,000 events were acquired with FACSDIVA6.1.3. Mean fluorescence intensity of the peptide binding to the cellswere determined using FlowJo 10.1. software (Tree Star; Ashland, Oreg.,USA). FACS analysis demonstrated that the synthetic peptides listed inSEQ ID NOs. 1-7 bind to the PBMC but at various intensities, confirmingthe ability of the synthetic peptides to bind to CD40 on mononuclearcells. Peptide 1 and Peptide 3 showed high affinity for binding tomononuclear cells whereas Peptide 7 has the least binding affinity tothe mononuclear cells.

Example 3. Specificity of Peptide Binding to CD40

In order to confirm the specificity of the peptides to interfere withCD40:CD154 interaction, the specificity of peptides 1-7 as listed intable 1 to bind to human CD154 by ELISA was tested. Briefly, 96 wellELISA (Nunc Immunolon) plates were coated with the peptides in carbonatebuffer. The wells are washed and probed with FLAG-tagged rhsCD40 (1μg/ml; Alexis) and developed with HRP conjugated rabbit anti-FLAGantibody. The plates were washed and the enzymatic activity wasdetermined using 3,3′,5,5′-tetramethyl-benzidine substrate for 15minutes and the reaction was stopped by the addition of 2 M H2SO4, andoptical density was measured at 450 nm, with the correction wavelengthset at 620 nm. Similar to the mononuclear cell binding studies, thePeptide #1 showed high binding affinity to recombinant human CD40,whereas Peptide 4 had the lowest binding affinity. These binding studiesindicate Peptide 2, Peptide 3, and Peptide 7 showed specific binding tohuman CD40, and can therefore block CD40-CD154 interaction.

Example 4. Co-Stimulation Blockade with Peptides AbrogatesDonor-Specific T and B Cell Proliferation

In order to determine the functional effect of co-stimulation blockadewith peptides targeting CD40, one-way mixed lymphocyte reactions (MLR)was performed. CF SE-labeled PBMC from 5 different RM were stimulatedwith irradiated PBMC for 6 days. Stimulation with PHA was used aspositive control. Initial screening with various concentration ofpeptides 1-7 listed in table 1 (15, 30 and 45 μM). These studiesdemonstrated that peptides added at 30 μM showed the maximum potentialfor costimulation blockade and this concentration used in furtherassays. The cells were incubated in the presence of peptides at 30 μM.Following stimulation, PBMC were stained with antibodies specific forCD4, CD8 and CD20 and proliferating cells were quantitated bydetermining the frequency of CF SE-low CD4, CD8 and CD20 cells. Stainedcells were acquired on a 3-laser BD Canto II instrument (BD Bioscience).A minimum of 200,000 events were acquired with FACSDIVA 6.1.3. FACSanalysis was performed with FlowJo Version 10 software. Analysis of theresults demonstrated that all the peptides 1-7 showed significantsuppression of CD4, CD8 and CD20 cell proliferation in 4/5 donorrecipient combinations. More significantly, Peptide 2 and Peptide 3suppressed the proliferation of CD4, CD8, and CD20 cells at a higherefficacy compared to the other peptides.

Example 5. Treatment of B Cells with Anti-CD40 Peptides Blocks B CellActivation

The ability of CD40-binding peptides 1-7 as listed in Table 1 to block Bcell activation was assessed by analyzing the expression of MHC class IImolecules on the B cell when subsequently incubated in the presence ofrecombinant hCD40L. Briefly, B cells were purified from 3 RM PBMC bymagnetic sorting. B cells were incubated with peptides at 30 μM for 15minutes at 37° C., then activated for 30 minutes with recombinanthCD40L, and then analyzed for expression of MHC class II by staining thecells with specific antibody. Stained cells were acquired on a 3-laserBD Canto II instrument (BD Bioscience). A minimum of 200,000 events wereacquired with FACSDIVA 6.1.3. FACS analysis was performed with FlowJoVersion 10 software. FACS analysis of the B cells demonstrated thatpre-incubation with the peptides resulted in a 50% reduction in MHCclass II expression on B cells following activation with CD40L.

This study identified new peptides that can bind to CD40 and inhibitCD40 activity (i.e., CD40 interaction with CD40L). The identifiedpeptides bind specifically to CD40. Peptide 2, Peptide 3, and Peptide 5listed in Table 1 exhibited a higher affinity to bind to CD40 whencompared to the control Peptide 1. When compared with Peptide 1; Peptide2, Peptide 3 and Peptide 5 show an enhanced ability to bind CD40 andinhibit CD40-CD40L interaction and down-stream signaling as evidenced bycomplete abrogation of T and B cell proliferation as well as activationof B cells.

Example 6. Treating Diabetes by Transplanting Islets and Providing aPreparatory Regimen Using CD40 Binding Peptide

This example examines effect of anti-CD40 peptides and the tolerogeniceffect of ECDI-treated donor cells for islet transplantation in vivo.The effect of an exemplary CD40 binding peptide, peptide 3 isillustrated. ECDI-treated splenocytes from a xenogeneic or allogeneicsource are administered to a human or a non-human primate transplantedwith islets, thereby minimizing the possibility of graft rejection inthe human or non-human primate. The preparatory regimen of this examplecan readily be adapted to allotransplantation or xenotransplantation inhuman recipients (for example, transplant of islets, kidneys, or othercells, tissues, or organs).

In an illustrative example, a streptozotocin-induced model of diabetesis utilized with non-human primate subjects. Diabetes is induced byintravenous treatment with streptozotocin. Recipient subjects aretransplanted with islets from a MHC-I disparate, one MHC-II DRB-allelematched donor. Recipients are treated with a short termimmunosuppressive regimen comprising: (i) an anti-CD40 peptide (Peptide3), given intravenously at a dose of 50 mg/kg on day −8, −1, 7, and 14relative to transplant; (ii) rapamycin, given orally from day −7 to day21 relative to transplant with a target trough level of 5-12 ng/mL;(iii) soluble TNF receptor, given intravenously at a dose of 1 mg/kg ondays −7 and 0 relative to transplant and subcutaneously at a dose of 0.5mg/kg on days 3, 7, 10, 14, and 21 relative to transplant; and (iv)antagonistic anti-IL-6 receptor antibody, given intravenously at a doseof 10 mg/kg on days −7, 0, 7, 14, and 21 relative to transplant.

Transplant recipients in experimental groups receive an intravenousinfusion of ECDI-treated apoptotic donor leukocytes (ADLs) together withanti-CD40 peptide, peptide 3 (or any one or more peptide selected frompeptides 1-17) before and optionally after transplantation. In somecases, ADLs together with an anti-CD40 peptide, peptide 3 (or any one ormore peptide selected from peptides 1-17) are administered about 8 daysbefore transplantation. In some cases, ADLs are administered about 7days before transplantation. In some cases, ADLs together with ananti-CD40 peptide, peptide 3 (or any one or more peptide selected frompeptides 1-17) are administered about 1 day after transplantation. Insome cases, ADLs together with an anti-CD40 peptide are administeredabout 7 days after transplantation. In some cases, ADLs together with ananti-CD40 peptide are administered about 14 days after transplantation.In some cases, ADLs together with an anti-CD40 peptide are administeredabout 7 or 8 days before transplantation, and about 1, 7, and/or 14 daysafter transplantation. ADLs can be from the same donor as the islets ora different donor as disclosed herein.

Transplant recipients in a control group do not receive ADLs. Smalldoses of exogenous insulin can be administered through day 21 aftertransplantation. Transplant recipients that receive ADLs together withan anti-CD40 peptide are expected to exhibit improved transplantsurvival compared to the group that do not receive ADLs. Transplantrecipients that receive ADLs together with an anti-CD40 peptide areexpected to exhibit improved rejection-free transplant survival comparedto the group that do not receive ADLs. Transplant recipients thatreceive ADLs together with an anti-CD40 peptide are expected to exhibitlong-term functional survival of islet allografts. For example,transplant recipients that receive ADLs together with an anti-CD40peptide are expected to exhibit improved blood glucose control aftertransplant (e.g., become normoglycemic), including after they stopreceiving exogenous insulin (e.g., past day 100 or day 365post-transplant). Blood glucose control can be evaluated, for example,by intravenous glucose tolerance test (IVGTT), a mixed meal tolerancetest (MMTT), or any other metabolic test established for monitoringpancreatic islet beta cell function. In IVGTT, exogenous glucose isinjected intravenously, and the blood glucose level is measured overtime after the injection. Transplant recipients that receive ADLstogether with an anti-CD40 peptide are expected to exhibit rapiddecreases in blood glucose levels and reduced area under the glucoseconcentration curve after IVGTT (e.g., comparable levels to those priorto streptozotocin treatment). Islet transplant recipients that receiveADLs together with an anti-CD40 peptide are expected to exhibitdecreased hemoglobin A1C levels after transplant. Transplant recipientsthat receive ADLs together with an anti-CD40 peptide are expected toexhibit increased C-peptide levels after transplant that are maintainedover time, indicating the transplanted islets are functional (e.g.,fasted C-peptide levels, glucose-stimulated C-peptide levels, and/orincrease in C-peptide levels upon glucose stimulation).

Example 7. Conjugation of Anti-CD40 Peptides to Mismatched ApoptoticDonor Leukocytes (ADLs) for Tolerance Induction

This example demonstrates that conjugating anti-CD40 peptides (e.g.,Peptides 1-17 listed in table 1) to apoptotic donor leukocytes (ADLs)can enhance the tolerance-inducing efficacy of a preparatory regimen. Inaddition, for donor-recipient pairs that are fully WIC class I and classII mismatched, additionally conjugating recipient-type WIC class IImolecules to ADLs can enhance ADL efficacy in inducing tolerance to atransplanted cell, tissue, or organ.

Coupling one or more peptides that can abrogate CD40-CD154 binding(e.g., Peptides 1-17 listed in table 1) to the surface of ADLs canenhance tolerance to a transplanted cell, tissue or organ, after uptakeof ADLs (for example, by recipient spleen marginal zone antigenpresenting cells or liven sinusoidal endothelial cells). Additionally,coupling recipient-type WIC class II peptides for presentation afteruptake of ADLs, derived from one (or more) of the transplant recipient'sWIC class II molecules can promote tolerance in the recipient, forexample, via regulatory T cell subsets.

Mixed Lymphocyte Reactions

To determine the ability of apoptotic donor leukocytes coupled via ECDIwith anti-CD40 peptides (e.g. peptides listed in Table 1), andrecipient-type DRB peptide to promote immune regulation andtransplantation tolerance, the following fully mismatched monkeyrecipient/donor pair and experimental conditions are selected to test amixed lymphocyte reaction (MLR) in vitro with WIC-defined stimulator(donor) and responder (recipient) peripheral blood mononuclear cells(PBMCs).

Recipient PBMCs are Mauritian Cynomolgus Monkey (Mafa M4A, M4A, M4B,M4B, M4DR, M1DR). Apoptotic donor leukocytes are Mauritian CynomolgusMonkey (M3A, M3A, M3B, M3B, M3DR, M3DR), with 14 μg of syntheticpeptides (e.g., Peptides 1-17 listed in table 1) conjugated to the cellsurface via ECDI.

In the MLR assay, 3×106 apoptotic donor leukocytes with and withoutconjugated anti-CD40peptide are used to stimulate 3×106 recipient PBMCsat 37° C. in a CO2 incubator. Serial samples collected post stimulationon day 1 and day 3 are analyzed for the induction of DC-10 (CD141+CD163+of CD14+CD16+), Treg cells (CD25hi CD127− FoxP3+ of CD4+) and Tr1 cells(CD49b+LAG-3+ of CD4+) by flow cytometry. Stimulated cells show that incomparison to PBLs stimulated with control apoptotic donor leukocytesalone, there is an increase in the frequency of Tregs on day 1 andfurther on day 3 following stimulation with anti-CD40 peptide-conjugatedapoptotic donor leukocytes. Similarly, a gradual increase in thefrequency of Tr1 cells (on day 1 and day 3), is observed followingstimulation with anti-CD40 peptide-conjugated versus control apoptoticdonor leukocytes. These results demonstrate that conjugating anti-CD40peptides (e.g., Peptides 1-17 listed in table 1) to apoptotic donorleukocytes can promote expansion of tolerance-promoting immuneregulatory cell subsets.

The preparatory regimen of this example can readily be adapted toallotransplantation or xenotransplantation in mammalian recipients (forexample, transplant of islets, kidneys, or other cells, tissues, ororgans, such as embryonic stem cell, induced pluripotent stem cell(iPS)-derived, or mesenchymal stem cell-derived cells, tissues andorgans).

In an illustrative example, in addition to anti-CD40 peptides (e.g.,Peptides 1-17 listed in table 1), recipient type MHC peptides areconjugated to ADLs, and the ADLs are used to induce tolerance to a humanislet transplant recipient.

Splenocytes are obtained from a fully MHC-I and MHC-II mismatchedxenogeneic or allogeneic source and are conjugated during ECDI treatmentto anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1). Followingthat, MHC class II chains, domains, and/or peptides are conjugatedduring ECDI treatment to generate ADLs conjugated to anti-CD40 peptidesand peptides derived from MHC-II. The ADLs conjugated to anti-CD40peptides and peptides derived from MHC-II are administered to humansubjects that receive islet transplants, thereby reducing thepossibility of graft rejection. Transplant recipients receive anintravenous infusion of ADLs conjugated to anti-CD40 peptides andpeptides derived from MHC-II before and optionally aftertransplantation, for example, on day −7 and day +1 relative totransplantation. ADLs can be from the same donor as the islets or adifferent donor as disclosed herein.

Recipient subjects are transplanted with islets from the fully MHC classI and MHC class II mismatched donor. Recipients are treated with a shortterm immunosuppressive regimen comprising: (i) an antagonistic anti-CD40peptide (e.g., Peptides 1-17 listed in table 1), given intravenously ata dose of 50 mg/kg on day −8, −1, 7, and 14 relative to transplant; (ii)rapamycin, given orally from day −7 to day 21 relative to transplantwith a target trough level of 5-12 ng/mL; (iii) soluble TNF receptor,given intravenously at a dose of 1 mg/kg on days −7 and 0 relative totransplant and subcutaneously at a dose of 0.5 mg/kg on days 3, 7, 10,14, and 21 relative to transplant; and (iv) antagonistic anti-IL-6receptor antibody, given intravenously at a dose of 10 mg/kg on days −7,0, 7, 14, and 21 relative to transplant.

Small doses of exogenous insulin can be administered through day 21after transplantation. Transplant recipients in control groups do notreceive ADLs, or receive ADLs without anti-CD40 peptide and MHC class IIpeptides conjugated. Transplant recipients that receive anti-CD40peptide and MHC-II peptides conjugated ADLs are expected to exhibitimproved survival compared to recipients that do not receive anti-CD40peptide and MHC-II-conjugated ADLs. Transplant recipients that receiveADLs conjugated with anti-CD40 peptide and peptides derived from MHC-IIare expected to exhibit improved rejection-free survival compared torecipients that do not receive anti-CD40 peptide and MHC-II-conjugatedADLs. Transplant recipients that receive ADLs conjugated to anti-CD40peptides and peptides derived from MHC-II are expected to exhibitlong-term functional survival of islet allografts compared to recipientsthat do not receive ADLs conjugated to anti-CD40 peptides and peptidesderived from MHC-II. For example, transplant recipients that receiveADLs conjugated to anti-CD40 peptides and peptides derived from MHC-IIare expected to exhibit improved blood glucose control after transplant(e.g., become normoglycemic), including after they stop receivingexogenous insulin (e.g., past day 100 or day 365 post-transplant). Bloodglucose control can be evaluated, for example, by intravenous glucosetolerance test (IVGTT), a mixed meal tolerance test (MMTT), or any othermetabolic test established for monitoring pancreatic islet beta cellfunction. In IVGTT, exogenous glucose is injected intravenously, and theblood glucose level is measured over time after the injection.Transplant recipients that receive ADLs conjugated to anti-CD40 peptidesand peptides derived from MHC-II are expected to exhibit rapid decreasesin blood glucose levels and reduced area under the glucose concentrationcurve after IVGTT (e.g., comparable levels to a healthy subject).Transplant recipients that receive ADLs conjugated to anti-CD40 peptidesand peptides derived from MHC-II are expected to exhibit decreasedhemoglobin A1C levels after transplant. Transplant recipients thatreceive anti-CD40L and MHC-II conjugated ADLs are expected to exhibitincreased C-peptide levels after transplant that are maintained overtime, indicating the transplanted islets are functional (e.g., fastedC-peptide levels, glucose-stimulated C-peptide levels, and/or increasein C-peptide levels upon glucose stimulation).

Example 8. Stem Cell-Derived B Cells Conjugated with Anti-CD40 Peptidefor Tolerance Induction to Cells, Tissues, or Organs Derived from theSame Stem Cell Donor

Stem cells from one donor can be differentiated into a first populationof cells for use as apoptotic donor leukocytes (ADLs), and separatelydifferentiated into a second population of cells for transplant. Thistechnique can be used to induce tolerance to any universal cell-derivedcell, tissue, or organ transplant.

The stem cells can be embryonic stems cells, induced pluripotent stemcells (iPSCs), and/or mesenchymal stem cells. The stem cells aredifferentiated into a first population of cells that express both MHCclass I and MHC class II antigens. For example, iPSCs from a transplantdonor are differentiated into B lymphocytes that express both MHC classI and II antigens. Methods of differentiating iPSCs into B cells aredescribed, for example, in French A et al. (2015), Stem Cells andDevelopment 24(9):1082-95. Preferably, for increased tolerogenicefficacy, the donor stem cell-derived B cells share one MHC class IIantigen with the recipient (e.g., at least one MHC class II DR allele,MHC class II DQ allele, or MHC class II DP allele). Separately, theiPSCs are differentiated into a second population of cells to betransplanted. The stem-cell derived B cells are treated with ECDI in thepresence of an anti-CD40 peptide (e.g., Peptides 1-17 listed in table 1)to generate anti-CD40 conjugated apoptotic donor leukocytes (anti-CD40peptide conjugated ADLs).

The stem cell-derived, anti-CD40 peptide conjugated ADLs areadministered to a subject that receives a transplant of cells, tissues,or organs derived from the same stem cell donor, thereby reducing thepossibility of transplant rejection. The transplant recipient receivesan intravenous infusion of stem cell-derived anti-CD40 peptideconjugated ADLs before and optionally after transplantation, forexample, on day −7 and day +1 relative to transplantation.

The recipients can optionally be treated with a short termimmunosuppressive regimen comprising: (i) an antagonistic anti-CD40peptide (e.g., Peptides 1-17 listed in table 1), given intravenously ata dose of 50 mg/kg on day −8, −1, 7, and 14 relative to transplant; (ii)rapamycin, given orally from day −7 to day 21 relative to transplantwith a target trough level of 5-12 ng/mL; (iii) soluble TNF receptor,given intravenously at a dose of 1 mg/kg on days −7 and 0 relative totransplant and subcutaneously at a dose of 0.5 mg/kg on days 3, 7, 10,14, and 21 relative to transplant; and (iv) antagonistic anti-IL-6receptor antibody, given intravenously at a dose of 10 mg/kg on days −7,0, 7, 14, and 21 relative to transplant.

The recipient is expected to exhibit improved rejection-free survivalcompared to the recipients that do not receive the stem-cell derived,anti-CD40 peptide conjugated ADLs. This technique can be used to inducetolerance to any universal cell-derived cell, tissue, or organtransplant.

Example 9. Dendritic and T Cell Immunomodulatory Effects of B-CellProducts Conjugated with Anti-CD40 Peptide

ECDI-fixed B cell products conjugated with CD40 binding peptide (e.g.,Peptides 1-17 listed in table 1) generated under different experimentalconditions are compared for their ability to induce maturation-arrest indendritic cells (DC). Human monocyte-derived DC are generated with IL-4and GCSF. These DC are incubated in the presence or absence of variousECDI-fixed B cell products coupled to inhibitors of DC maturation (e.g.,rapamycin, curcumin, vitamin D3, Bay-117085, siCD40, cobaltprotoporphyrin, and α1-antitrypsin). Readouts of DC maturation arrestinclude i) expression of DC phenotypic markers (CD83, CD80, CD86, MHCclass II, CD40), ii) STAT-6 phosphorylation, iii) RELP nucleartranslocation, iv) IL-12p70 production, v) allostimulatory capacity, andvi) priming of T cells with regulatory phenotypes(CD4+CD25hiCD127lowFoxp3+ Tregs and CD4+CD49b+Lag-3+CD45RA− Tr1 cells).ECDI-treated B cell products are identified that inducematuration-arrest in DC.

The effects of ECDI-fixed donor B lymphocytes on immune profiles inresponder peripheral blood lymphocytes (PBL) is evaluated. ECDI-fixeddonor B cells are generated, including B cells with various anti-CD40peptides conjugated to their surface by ECDI (e.g., Peptides 1-17 listedin table 1). One-way mixed lymphocyte reactions (MLRs) are performedusing PBL from fully mismatched, one DRB-matched, or one-DQ matcheddonor-recipient pairs, with or without the addition of increasing dosesof ECDI-fixed anti-CD40 peptide conjugated donor B cells.

Cells are phenotyped and proliferation evaluated at various time pointsby multi-parametric flow cytometry (e.g., including CFSE dilution andstaining for markers that differentiate cell subsets of interest).Blocking antibodies are added and distinct cell subsets are depleted todissect underlying mechanisms and to determine how the enhancedECDI-fixed B cell products influence T cell immunity (e.g., B cellproducts with or without coupled anti-CD40 peptides). Readouts includei) fold-proliferation of CD4+ and CD8+ T cells with effector phenotypesas determined by surface markers, intracellular cytokines, and/ortranscription factors; ii) fold-proliferation of CD4+ and CD8+ T cellswith regulatory phenotypes as determined by surface markers,intracellular cytokines, and/or transcription factors; and iii) CD8+ Tcell-mediated cytotoxicity against target cells in the MLR.

Example 10. Efficacy of Peri-Transplantation Infusions of Ex VivoExpanded ADLs Coupled to Anti-CD40 Peptides, in Inducing Donor SpecificTolerance to Kidney Allograft

The efficacy of peri-transplantation infusions of ADL products coupledto anti-CD40 peptides (e.g., Peptides 1-17 listed in table 1), forinducing stable renal allograft tolerance is evaluated in oneDRB-matched, SI non-human primates. An experimental group receives ADLproducts comprising ex vivo expanded donor B cells coupled to anti-CD40peptides (e.g., Peptides 1-17 listed in table 1), while a control groupreceives saline infusions. Group sizes of n=5 are studied; up to 2additional non-human primates are added per group to replacenon-informative recipients (e.g., recipients that contract unrelateddiseases). SI mammalian recipients are identical in both groups andlong-term maintenance drugs are not given to any recipient. Renalallograft failure is defined by serum creatinine>2.5 mg/dL and confirmedby graft histology.

Purpose-bred, qualified non-human primate donors and recipients (exam,labs, microbial screen, vaccination, etc.) are selected from qualifiedvendors. The donors and recipients have a defined MHC disparity (MHCclass I-disparate and one MHC class II DRB allele-matcheddonor-recipient pairs, based on high-resolution MHC class I and IIgenotyping using Fluidigm Access Arrays to generate amplicons for deepsequencing).

Recipients with evidence of existing allo-reactive memory can beexcluded from the study. Eligibility criteria for recipients can includeABO compatibility, low memory alloreactivity as defined by negativepanel reactive antibodies (PRA; OneLambda Bead assay), negativedonor-specific antibodies (DSA) by flow, and IFN-γ ELISPOT≤12 SFC/106PBMC (B cell ELISPOT) against donor non-human primate. Male or femalenon-human primate recipients are trained for cooperation andinstrumented with indwelling central and intraportal vascular access.

Kidney transplantation in non-human primates follows establishedprocedures. Briefly, following systemic heparinization of both donor andrecipient, the donor organ is excised and the vessels are anastomosed tothe recipient's infrarenal aorta and vena cava. Typically, this isperformed in a left-to-right fashion owing to the longer length of theleft renal vessels. The donor ureter is tunneled through theretroperitoneum and a primary ureteroneocystostomy is formed typicallyon the posterior wall of the bladder using a modifiedLeadbetter-Politano approach. Particular attention is paid to avoidurine leakage and ureteral stenosis. Bilateral native nephrectomy iscompleted prior to closure.

Ex vivo expanded and ECDI-fixed donor B cells coupled to anti-CD40peptides (e.g., Peptides 1-17 listed in table 1) are infused IV intoexperimental recipients on days −7 and +1 at a dose of 0.25×109/kgrecipient body weight. Approximately 60 ml of blood (corresponding to 1%of body weight) is drawn from donors on day −21 or −22 (±2 days)relative to planned renal transplant, and B cells are purified bymagnetic sorting using non-human primate CD20 beads. Alternatively oradditionally, B cells can be enriched from leukapheresis products. A Bcell expansion protocol is adapted from the culture system reported bySu et al (J Immunol 2016, 197:4163-76). Purified B cells (approximately24×106 B cells from 60 ml of blood) are expanded ex vivo in a GREX100Mflask (Wilson Wolf) until day −7 in RPMI 1640 medium with added 5%rhesus macaque serum, 55 μM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin,100 μg/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 1% MEMnonessential amino acids. The culture medium is supplemented withanti-CD40 peptides (e.g., Peptides 1-17 listed in table 1), IL-2 (50ng/ml), IL-4 (10 ng/ml), IL-21 (10 ng/ml), and BAFF (10 ng/ml). Inputcell numbers, medium volume, and the concentration of CD40L-multimericare optimized in feasibility studies. Cells are counted after 7 and 14days, split after 14 days (day −7), and the cells not infused on day −7are expanded for another 8 days for infusion on day +1. On the day ofinfusion, cells are agitated on ice for 1 hour with ECDI (30 mg/mL per3.2×108 cells) in DPBS in the presence of the CD40 binding peptides(e.g., Peptides 1-17 listed in table 1), washed, cleaned to removenecrotic cells and microaggregates, and assessed for viability/necrosisby AO/PI fluorescent microscopy. ECDI-fixed B cells coupled to CD40binding peptides, meeting all release criteria, are loaded into coldsyringes for IV infusion with a maximum concentration of 20×106cells/mL; the cells remain on ice until recipient administration.Induction of apoptosis is monitored in vitro by incubating an aliquot ofECDI-fixed cells coupled to CD40 binding peptides at 37° C. for 4-6hours, labelling with Annexin V/PI, and analyzing via fluorescentmicroscopy or flow cytometry.

Identical short-term immunosuppression and anti-inflammatory therapiesare administered to control and experimental subjects. The first dose ofeach drug is given on day −8 or −7 relative to transplantation on day 0.The antagonistic anti-CD40 peptides are given intravenously at 50 mg/kgon days −8, −1, 7, and 14. Rapamycin (Rapamune®) is given orally (PO)from day −7 through day 21 post-transplant; the target trough level is 5to 12 ng/ml. Concomitant anti-inflammatory therapy is with i) α-IL-6R(tocilizumab, Actemra®) at 10 mg/kg IV on days −7, 0, 7, 14, and 21, andii) sTNFR (etanercept, Enbrel®) at 1 mg/kg IV on days −7 and 0 and 0.5mg/kg subcutaneously on days 3, 7, 10, 14, and 21.

The primary efficacy outcome is the proportion of transplanted non-humanprimate with rejection-free allograft survival (confirmed byhistopathology) at day 365 post-transplant. Accordingly, follow-up is today 365 or graft failure, whichever occurs first. The group experimentalgroup that received ADLs coupled to CD40 binding peptides is expected toexhibit enhanced rejection-free allograft survival compared to thecontrol group that received only the short-term immunosuppression andanti-inflammatory therapies.

1. A method of treating inflammation in a subject, the methodcomprising: administering to the subject an effective amount of apharmaceutical composition that comprises: an isolated peptide thatcomprises an amino acid sequence with at least 90% sequence identity toany one of SEQ ID NOs: 1-6 or SEQ ID NOs: 8-17, as determined by BLASTalgorithm; and a pharmaceutically acceptable excipient, carrier, ordiluent, wherein said administering is in an amount sufficient to resultin a reduction of B cell activation in said subject as compared to the Bcell activation in said subject prior to said administering, therebytreating the inflammation in the subject.
 2. The method of claim 1,wherein said isolated peptide comprises an amino acid sequence set forthin any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 8-17.
 3. The method ofclaim 1, wherein said isolated peptide further comprises at least onemodification, wherein said at least one modification is a chemicalmodification, or a post-translational modification.
 4. The method ofclaim 3, wherein said chemical modification is selected from a groupconsisting of ubiquitination, pegylation, lipidation, glycosylation,alkylation, or thiolation.
 5. The method of claim 3, wherein saidpost-translational modification is an acetylation, acylation,ADP-ribosylation, amidation, carboxylation, hydroxylation, disulfidebond formation, glycosylation, phosphorylation, proteolytic processing,sulfation, methylation, acyl lipidation, prenylation, methylation, ormyristoylation.
 6. The method of claim 1, wherein said isolated peptideis cyclized.
 7. The method of claim 1, wherein said isolated peptide isfurther conjugated to a carrier polypeptide, a detectable agent, apeptide tag, a magnetic particle, a diagnostic agent, a therapeuticagent, a nanoparticle, or a combination thereof.
 8. The method of claim1, wherein said isolated peptide is formulated in a liposome or ananoparticle delivery system.
 9. The method of claim 1, wherein saidisolated peptide inhibits binding of a CD40 protein to a CD154 protein.10-20. (canceled)
 21. The method of claim 1, wherein said inflammationis associated with an autoimmune disease.
 22. A method of reducingproliferation of one or more of T cells and B cells in a subject, themethod comprising: administering to the subject an effective amount of apharmaceutical composition that comprises an isolated peptide thatcomprises an amino acid sequence with at least 90% sequence identity toany one of SEQ ID NOs: 1-6 or SEQ ID NOs: 8-17, as determined by BLASTalgorithm and a pharmaceutically acceptable excipient, carrier, ordiluent, wherein the pharmaceutical composition is administered in anamount sufficient to result in inhibition of proliferation of at leastone of T cells and B cells in said subject as compared to inhibition ofproliferation of T cells and B cells in said subject prior to saidadministering, thereby treating the inflammation in the subject.
 23. Themethod of claim 22, wherein said isolated peptide comprises an aminoacid sequence set forth in any one of SEQ ID NOs: 1-6 or SEQ ID NOs:8-17.
 24. The method of claim 22, wherein said isolated peptide furthercomprises at least one modification, wherein said at least onemodification is a chemical modification, or a post-translationalmodification.
 25. The method of claim 22, wherein said isolated peptideis cyclized.
 26. The method of claim 22, wherein said isolated peptideis formulated in a liposome or a nanoparticle delivery system.
 27. Themethod of claim 22, wherein said isolated peptide inhibits binding of aCD40 protein to a CD154 protein.
 28. A method of treating a neoplasticdisease characterized by CD40 expression in a subject, the methodcomprising: administering to the subject an effective amount of apharmaceutical composition that comprises an isolated peptide thatcomprises an amino acid sequence with at least 90% sequence identity toany one of SEQ ID NOs: 1-6 or SEQ ID NOs: 8-17, as determined by BLASTalgorithm and a pharmaceutically acceptable excipient, carrier, ordiluent, wherein the pharmaceutical composition is administered in anamount sufficient to result in a reduction of CD40 expression in saidsubject as compared to CD40 expression in said subject prior to saidadministering, thereby treating the neoplastic disease characterized bysaid CD40 expression in said subject.
 29. The method of claim 28,wherein the isolated peptide blocks CD40-CD40L signaling.
 30. The methodof claim 28, wherein the neoplastic disease is a lymphoma, a myeloma ora carcinoma.